System and Methods for Making and Processing Emulsions

ABSTRACT

An automated template bead preparation system is provided and includes a membrane-based emulsion generation subsystems, a thermal plate and subsystem, and a continuous centrifugation emulsion breaking and templated bead collection subsystem. The emulsion generation subsystem provides uniformity in the preparation of an inverse emulsion and may be used to create large or small volume inverse emulsions rapidly and reproducibly. An emulsion-generating device is provided that can supply a continuous stream of an inverse emulsion to a thermal subsystem, in automated fashion. The thermal subsystem can treat an inverse emulsion passed therethrough. The continuous centrifugation subsystem can continuously break a thermally cycled inverse emulsion and collect template beads formed in the aqueous microreactor droplets of the inverse emulsion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/656,638, filed Jun. 7, 2012 and entitled “SYSTEM AND METHODS FORMAKING AND PROCESSING EMULSIONS,” which is incorporated herein byreference in its entirety.

This application claims benefit of U.S. Provisional Application No.61/671,481, filed Jul. 13, 2012 and entitled “SYSTEM AND METHODS FORMAKING AND PROCESSING EMULSIONS,” which is incorporated herein byreference in its entirety.

This application is a continuation-in-part application of U.S.Non-Provisional application Ser. No. 13/442,547, filed Apr. 9, 2012 andentitled “SYSTEM AND METHODS FOR AUTOMATED SAMPLE LIBRARY PREPARATION,”which claims benefit of U.S. Provisional Application No. 61/472,869,filed Apr. 7, 2011 and entitled “SYSTEM AND METHODS FOR AUTOMATED SAMPLELIBRARY PREPARATION,” claims benefit of U.S. Provisional Application No.61/489,928, filed May 25, 2011 and entitled “SYSTEM AND METHODS FORMAKING AND PROCESSING EMULSIONS,” and claims benefit of U.S. ProvisionalApplication No. 61/583,079, filed Jan. 4, 2012 and entitled “SYSTEM ANDMETHODS FOR MAKING AND PROCESSING EMULSIONS,” each of which isincorporated herein by reference in its entirety.

FIELD

The present teachings relate to devices, systems, and methods forpreparing and reacting within emulsions, including emulsions useful inbiological reaction processes, for example, useful in a polymerase chainreaction (PCR).

INTRODUCTION

A number of biological sample analysis methods rely on samplepreparation steps as a precursor to carrying out the analysis methods.For example, a precursor to performing many biological sequencingtechniques (e.g., sequencing of nucleic acid) includes amplification ofnucleic acid templates in order to obtain a large number of copies(e.g., millions of copies) of the same template.

Polymerase chain reaction is a well understood technique for amplifyingnucleic acids which is routinely used to generate sufficiently large DNApopulations suitable for downstream analysis. Recently, PCR-basedmethods have been adapted to amplifying samples contained withinemulsions for sequencing applications. In such amplification methods aplurality of biological samples (e.g. nucleic acid samples) may beindividually encapsulated in microcapsules of an emulsion and PCRamplification conducted on each of the plurality of encapsulated nucleicacid samples simultaneously. Such microcapsules are often referred to as“microreactors” because the amplification reaction occurs within themicrocapsule.

In some cases, the microcapsule can include a template bead, alsoreferred to as a P1 bead or a primer 1 bead and the amplificationprocess may be referred to as bead-based emulsion amplification, forexample, as described in US 2008/0003571 A1 to McKernan et al., which isincorporated herein in its entirety by reference. In such a technique,beads along with DNA templates are suspended in an aqueous reactionmixture and then encapsulated in an inverse (water-in-oil) emulsion. Thetemplate DNA may be either bound to the bead prior to emulsification ormay be included in solution in the amplification reaction mixture. Forfurther details regarding techniques for bead emulsion amplification,reference is made to PCT publication WO 2005/073410 A2, entitled“NUCLEIC ACID AMPLIFICATION WITH CONTINUOUS FLOW EMULSION,” whichpublished internationally on Aug. 11, 2005, and is incorporated byreference in its entirety herein.

SUMMARY

According to various embodiments of the present teachings, amplified DNAfragments tethered to a particle or bead can be prepared. Device,systems, apparatuses, and methods are described herein relating to theamplified polynucleic acid tethered particles or beads.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate exemplary embodiments andtogether with the description, serve to explain various principles. Theskilled artisan will understand that the drawings, described below, arefor illustration purposes only. The drawings are not intended to limitthe scope of the present teachings in any way.

FIG. 1 is a flow diagram showing the method steps involved according tovarious embodiments of the present teachings.

FIG. 2 is a perspective view of an automated sample preparation systemfor conducting automated sample preparation according to variousembodiments of the present teachings.

FIG. 3 depicts a schematic diagram and side view of a membrane-basedemulsion-generating system for generating emulsion droplets, accordingto various embodiments of the present teachings.

FIG. 4A is a cross-sectional view showing the details of amembrane-based emulsion-generating device according to variousembodiments of the present teachings.

FIG. 4B is a top view of the device shown in FIG. 4A.

FIG. 4C is a bottom view of the device shown in FIG. 4A.

FIG. 4D is an exploded view of the internal components of the deviceshown in FIG. 4A.

FIG. 5 is an exploded view of the internal components of amembrane-based emulsion-generating device according to yet otherembodiments of the present teachings.

FIG. 6A is a side perspective view of a thermocycling subsystem inaccordance with various embodiments of the present teachings.

FIG. 6B is an exploded view of the thermocycling subsystem shown in FIG.6A.

FIG. 6C is a side perspective view another thermocycling subsystem inaccordance with various embodiments of the present teachings.

FIG. 6D is an exploded view of the thermocycling subsystem shown in FIG.6C.

FIG. 7A is a perspective view of a heating subassembly for use in athermocycling subsystem in accordance with various embodiments of thepresent teachings.

FIG. 7B is a perspective view and mirror image of the heatingsubassembly shown in FIG. 7A.

FIG. 8A is a perspective view of a thermocycling plate for use in athermocycling subsystem in accordance with various embodiments of thepresent teachings.

FIG. 8B is a rear perspective view of the thermocycling plate shown inFIG. 8A.

FIG. 8C is a perspective view and mirror image of the thermocyclingplate shown in FIG. 8A.

FIG. 8D is a rear perspective view of the thermocycling plate shown inFIG. 8C in accordance with various embodiments of the present teachings.

FIG. 9A is a perspective view of a bottom region of a fluid passagefound in a thermocycling plate in accordance with various embodiments ofthe present teachings.

FIG. 9B is a perspective view of another bottom portion of a fluidpassage that can be found in a thermocycling plate in accordance withvarious embodiments of the present teachings.

FIG. 10A is a perspective view of a gasket that can be used incombination with a thermocycling subsystem in accordance with variousembodiments of the present teachings.

FIG. 10B is a perspective view of the gasket shown in FIG. 10A incombination with a thermocycling subsystem in accordance with variousembodiments of the present teachings.

FIG. 11A is a perspective view of an emulsion PCR system showing athermocycling subsystem in an open configuration in accordance withvarious embodiments of the present teachings.

FIG. 11B is a perspective view of an emulsion PCR system showing athermocycling subsystem in a closed configuration in accordance withvarious embodiments of the present teachings.

FIG. 12A is a front perspective view of a centrifuge subsystem inaccordance with various embodiments with the present teachings.

FIG. 12B is a side perspective view of the centrifuge subsystem shown inFIG. 12A.

FIG. 12C is a rear perspective view of the centrifuge subsystem shown inFIG. 12A.

FIG. 12D is a bottom perspective view of the centrifuge subsystem shownin FIG. 12A.

FIG. 13A is a side perspective view of the centrifuge subsystem shown inFIG. 12A, in an open configuration.

FIG. 13B is another side perspective view of the centrifuge subsystemshown in FIG. 12A, in an open configuration.

FIG. 14A is a side perspective view of a collection tube in accordancewith various embodiments of the present teachings.

FIG. 14B is another side perspective view of the collection tube shownin FIG. 14A.

FIG. 14C is a top perspective of the collection tube shown in FIG. 14A.

FIG. 15 is a plan view of a fluid distribution device (slinger) inaccordance with various embodiments of the present teachings.

FIG. 16 is a top perspective view of the centrifuge subsystem in an openconfiguration of the centrifuge subsystem shown in FIG. 12A.

FIG. 17 is a perspective view of a peripheral gutter for use in acentrifuge subsystem as shown in FIG. 12 A.

FIG. 18A is a top perspective of the centrifuge subsystem as shown inFIG. 12A with showing the collection tube, slinger, and peripheralgutter components.

FIG. 18B is a side perspective view of the centrifuge subsystem assemblyshown in FIG. 18A.

FIG. 18C is a cross sectional view of the centrifuge subsystem assemblyas shown in FIG. 18A.

FIG. 19 is a perspective view of a centrifuge subsystem operablyconnected with a source fluid and source fluid pump in accordance withvarious embodiments of the present teachings.

FIG. 20 is a flow chart depicting a method of centrifugation andcollection of a sample in accordance with various embodiments of thepresent teachings.

FIG. 21A is a perspective view of a plate-based emulsion-generatingdevice according to various embodiments of the present teachings.

FIG. 21B is a cross-sectional view taken along line B-B of FIG. 21A.

FIG. 22 is a cross-sectional side view of a plate-basedemulsion-generating device according to various embodiments of thepresent teachings.

FIG. 23 is perspective, partial cutaway view of a plate-basedemulsion-generating device according to various embodiments of thepresent teachings.

FIGS. 24A-24F include illustrations of exemplary thermocycling platedesigns.

FIG. 25, FIG. 26, FIG. 27, FIG. 28, FIG. 29, FIG. 30, and FIG. 31include illustrations of exemplary emulsion-generating devices.

FIG. 32 and FIG. 33 include illustrations of exemplary channel gasketdesigns.

FIG. 34 and FIG. 35 include illustrations of exemplary thermocyclingplates.

FIG. 36 and FIG. 37 include illustrations of exemplaryemulsion-generating devices.

FIG. 38 includes an illustration of an exemplary adapter to interfacewith a centrifuge.

FIG. 39 and FIG. 40 include illustrations of exemplary thermocyclingplates.

DESCRIPTION

According to various embodiments of the present teachings, amplified DNAfragments tethered to a particle or bead can be prepared. The method canbegin by forming an inverse emulsion comprising a plurality of aqueousdroplet microreactors encapsulated and separated from one another by acarrier fluid, for example, an immiscible oil or a fluorinated liquid.Each microreactor, or at least one of them, can contain a template bead,also referred to as a P1 bead or a primer 1 bead, and PCR ingredients.The amplification process may be referred to as a bead-based emulsionamplification, for example, as described in U.S. Patent ApplicationPublication No. US 2008/0003571 A1 to McKernan et al., which isincorporated herein in its entirety by reference. In such a technique,beads along with DNA templates are suspended in an aqueous reactionmixture (a microreactor mixture) and then encapsulated by the immiscibleliquid in an inverse (water-in-oil) emulsion. The template DNA may beeither bound to the bead prior to emulsification or may be included insolution in the amplification reaction mixture. For further detailsregarding techniques for bead emulsion amplification, reference is madeto PCT publication No. WO 2005/073410 A2, entitled “NUCLEIC ACIDAMPLIFICATION WITH CONTINUOUS FLOW EMULSION,” which publishedinternationally on Aug. 11, 2005, and is incorporated by reference inits entirety herein.

According to various embodiments, a method and system are provided forautomated sample preparation for sequencing applications. In someembodiments, bead-based emulsion amplification is performed uponformation of an emulsion which encapsulates a template DNA strand, abead upon which amplicons formed from the template DNA strand areretained, and a reagent mixture for supporting the amplificationreaction and providing ingredients therefore. The emulsion can comprisean inverse (water-in-oil) emulsion with the aqueous phase (e.g., themicroreactor mixture) including the reagent mixture and the bead, andthe carrier fluid including oil or other non-aqueous liquid that ispartially or completely immiscible with the aqueous phase.

Particle-based, e.g., bead-based, emulsion amplification can beperformed using an emulsion which encapsulates a template DNA strand, abead upon which amplicons formed from the template DNA strand areretained, and a reagent mixture for supporting the amplificationreaction and providing ingredients therefore. The emulsion can comprisean inverse (water-in-oil) emulsion with the aqueous phase (e.g., themicroreactor mixture) including the reagent mixture and the bead, andthe carrier fluid including oil or other non-aqueous liquid that ispartially or completely immiscible with the aqueous phase.

According to various embodiments, an emulsion amplification system isprovided. The amplification system can comprise a first sample reactionplate, as described herein, and a heating subassembly, as describedherein. The heating subassembly of the amplification system can alsocomprise a complementary heating block as described herein. The heatingsubassembly of the amplification system can also comprise a secondsample reaction plate. The amplification system can be insulated usingany means or mechanism. For example, a gasket can be used for insulatingpurposes.

In some embodiments, a sample reaction plate is provided in accordancewith the present teachings. The sample reaction plate generally maycomprise a slab housing and a main fluid passage passageway that passesthrough the slab housing. The main fluid passageway is disposed in theslab housing and can include an inlet, an outlet, and various fluidpassage segments in fluid communication with adjoining fluid passages.The main fluid passageway can comprise a number of fluid passagescollectively in fluid communication. Such fluid passages can include aninitial fluid passage in fluid communication with the inlet, atransition fluid passage in fluid communication with the initial fluidpassage, main fluid passage in fluid communication with the transitionfluid passage. The main fluid passage can be in direct fluidcommunication with the outlet or via an exit fluid passage. The mainfluid passage, the initial fluid passage, or both can have a tortuousshape. The main fluid passage, the initial fluid passage, or both canhave a plurality of repeating segments or repeats (fluidic paths)between their respective partitions.

A heating subassembly is also provided by the present teachings and canbe used in combination with one or more sample reaction plates describedherein. The heating subassembly can comprise a first heating block, afirst heat control unit, a second heat control unit, and, optionally, anegative load device. The first heating block can comprise a firststatic heating zone, a heating zone partition, and a second staticheating zone separated from the first static heating zone by the heatingzone partition. The first heat control unit is operably associable withthe first static heating zone. The second heat control unit is operablyassociable with the second static heating zone. In various embodiments,the first temperature or temperature range, or the second temperature ortemperature range, can be a temperature or temperature range sufficientto allow for denaturing of double-stranded nucleic acid, annealing ofnucleic acids, or extension of nucleic acids, or any combinationthereof. While a single heating block can be employed in the heatingsubassembly, use of a second, complementary heating block can provideadditional temperature control and is particularly advantageous when twosample reaction plates are used. Accordingly, the heating subassembly ofthe present teachings can comprise a complementary heating block ormated heating blocks.

In some embodiments, sample amplification is provided by thermocyclingin accordance with the present teachings. The method can comprise one ormore of the following steps, the order of which can be varied, orwherein one or more of the steps can be repeated. A sample fluid can bepassed through a sample reaction plate comprising a plurality ofregions. A hot start region corresponding to an initial fluid passage ofthe sample reaction plate can be heated and a denaturation regioncorresponding to a portion of a main fluid passage proximal a secondpartition of the sample reaction plate can be heated to a firsttemperature or temperature range. An annealing/extension regioncorresponding to a portion of the main fluid passage proximal a thirdpartition of the sample reaction plate can be heated to a secondtemperature or temperature range. The sample fluid passed through thefluid passageway can comprise a water-in-oil emulsion comprising aplurality of aqueous polymerase chain reaction (PCR) reaction droplets.

In accordance to various embodiments of the present teachings, acentrifuge system is provided that is suitable for integration into anemulsion amplifying system or equivalent device. The centrifuge cancomprise a centrifuge housing, a motor comprising a rotor axle mountedin the housing motor aperture, and a rotor mounted on the rotor axle.

A fluid collection tube is provided that can comprise a main tube bodyand a tube extension. The main tube body can comprise a main bodysidewall surrounding a tube interior with a tube opening at a first end,and a second end distal to the first end providing a sealed base. Thetube extension can comprise a tube extension sidewall defining a fluidexit channel in fluid communication with the tube interior through atube channel inlet proximal to the tube opening and extending to a tubechannel outlet distal to the tube opening.

A fluid distribution device, also referred to herein as a “slinger,” isprovided by the present teachings. The slinger can comprise sidewallsdefining a central channel comprising a first end, a central zone, and asecond end along a longitudinal axis. Sidewall lateral extensions, alsoreferred to as “wings” herein, can be provided extending away from thesidewalls on either side of the central channel. The wings are useful inmating with and ensuring a secure connection with a fluid distributiondevice receptacle of a centrifuge rotor. Rather than use aninsertable/detachable slinger, a slinger can be employed that ispermanently or integrally associated with the rotor housing.

A centrifuge rotor is provided by the present teachings, which isparticularly suitable for separating water-in-oil emulsions and removingthe oil phase of such an emulsion. The centrifuge rotor can comprise arotor housing having a bisecting rotor axis perpendicular to a centralrotor axis, a rotor basin formed by the rotor housing, a rotor basinfloor, and a rotor basin sidewall lining the rotor housing basin andextending up toward a rotor top rim having an inner perimeter and anouter perimeter. The rotor can further comprise at least one collectiontube receptacle comprising an opening formed in the basin sidewall, andat least one tube extension receptacle having a grove formed in therotor top rim and extending from the inner perimeter to the outerperimeter. The rotor can also comprise at least one liquid distributiondevice (slinger) receptacle extending from the rotor basin floor andhaving a distribution device receptacle longitudinal axis.

A method of recovering a polymerase chain reaction (PCR) product from awater-in-oil emulsion is provided in accordance with the presentteachings. The method can be performed using the centrifuge orcentrifuge components of the present teachings.

The method can comprise one or more of the following steps. At least onecollection tube can be filled with an aqueous solution and inserted intoa tube receptacle of a centrifuge rotor. At least one fluid distributiondevice (slinger) can also be inserted into a fluid distribution devicereceptacle of the centrifuge rotor. Sample fluid, which can comprise aPCR product in a water-in-oil emulsion, can be fed into the fluiddistribution device. The centrifuge rotor is spun to deliver the samplefluid to the at least one collection tube, and the emulsion is partly orcompletely broken in the at least one collection tube. The centrifugerotor motor can also be spun to remove an oil phase from the collectiontube. The method can further comprise recovering PCR product from the atleast one collection tube.

As shown in FIG. 1, automated bead sample preparation method is providedthat can comprise generating an emulsion in a first step 50, conductingemulsion PCR in a second step 54, and conducting an emulsion breakingand bead washing third step 58. According to various embodiments, anemulsion can be generated that comprises droplets of an aqueous phase,herein called microreactors, in which clonal amplification can takeplace. Microreactors containing a single template bead and a singletemplate, called monoclonal microreactors, are desired and can be formedaccording to the present teachings. Some microreactors, however, can bepolyclonal such that they contain multiple templates, non-clonal suchthat they contain no template, or multi-bead-containing Somemicroreactors can exhibit a combination of these undesirable features.

In some embodiments, microreactors can be formed by mixing together aplurality of template beads, a library of templates from a sample, DNApolymerase, a buffer, dNTPs, one or more surfactants, and a pair ofprimers, in an aqueous phase solution to form an aqueous phase. Theaqueous phase can then be contacted with an oil phase and emulsified toform an inverse emulsion comprising the microreactors in the oil phase.Although reference is made herein to an oil phase, it is to beunderstood that other PCR-compatible immiscible liquids can be used forthe continuous phase of the inverse emulsion provided the liquid isimmiscible with the aqueous phase. Fluorinated compounds, fluorinatedliquids, minerals oils, silicone oils, and the like, can be used in someembodiments.

In some embodiments, microreactors can be formed by including at leastone additive. For example, an additive includes a polymer compoundcomprising a homo-polymeric or hetero-polymeric compound. In someembodiments, a polymer compound includes amphiphilic and hydrophiliccompounds. In some embodiments, a polymer compound includes syntheticand naturally-occurring compounds. In some embodiments, a polymercompound can be soluble in water, alcohol, ether, ketone or ester. Insome embodiments, a polymer compound comprises a chain of two or moretetrahydrapyrrole monomers. In some embodiments, a tetrahydrapyrrolemonomer comprises a five-membered heterocyclic ring. In someembodiments, in a chain of tetrahydropyrrole rings, one or moretetrahydropyrrole rings comprise a nitrogen atom reacted with a carbonylor carboxylic acid compound. In some embodiments, a polymer compoundincludes but is not limited to polyvinylpyrrolidone (e.g., povidone orcrospovidone), poly(4-vinylphenol), and vinylpyrrolidone/vinyl acetatecopolymer (e.g., copovidone). In some embodiments, a polymer compoundcomprises two or more monomers of N-vinyl-pyrrolidone, includingmodified polymers thereof. Modified polymers ofpoly(N-vinyl-pyrrolidone) comprise monofunctionalized (e.g., hydroxyl orcarboxy end group) polymers, side-chain conjugates (e.g., poly- andmultifunctional side chains), and grafted copolymers. In someembodiments, polyvinylpyrrolidone includes a range of molecular weightpolymers including 2500-750,000 g/mol. In some embodiments,polyvinylpyrrolidone includes various molecular weight polymersincluding average molecular weights of about 5 kD-55 kD, for exampleaverage molecular weights of about 10 kD, 29 kD, 40 kD, and 55 kD. Insome embodiments, polyvinylpyrrolidone can be commercially-available,including: Kollidon, Luviskol, Albigen A, Devergan (BASF), PVP (GeneralAnaline and Film, Corp.), PVP 10 kD (Sigma-Aldrich, #PVP10-100G),Plasdone (General Analine and Film, Corp.), Collacral, Luviskol VA, andPVP/VA (copolymers of vinyl acetate, General Analine and Film, Corp.).Alternatively, the components and solutions can be free of PVP.

In some embodiments, a library of nucleic acid templates can beamplified in a microreactor. In some embodiments, the yield or reactionrate of a nucleic acid amplification reaction can be improved byaddition of a polymer compound. For example, a polymer compound canserve as a molecular crowding agent in a nucleic acid amplificationreaction, or a polymer compound can bind or neutralize polar moleculesor polyphenolic compounds that may interfere with a nucleic acidamplification reaction. In some embodiments, a polymer compound can bindor neutralize a polyether compound including oligomers or polymers ofethylene oxide (e.g., polyethylene glycol). In some embodiments, apolyvinylpyrrolidone can bind a polyethylene glycol. In someembodiments, a polymer compound can bind polyethylene glycol having amolecular weight of less than about 20,000 g/mol, or more than about20,000 g/mol.

In another example, a polymer compound can serve as a surfactant. Asurfactant can stabilize an oil and aqueous mixture. A surfactant canreduce surface tension at an interface between oil and aqueous phases. Asurfactant can reduce loss of enzymes (e.g., polymerase) to an oil-waterinterface which can make the enzymes available to catalyze a nucleicacid amplification reaction.

In some embodiments, the yield of nucleic acids deposited onto a surfaceor into a well can be improved by inclusion of a polymer compound. Forexample, one or more beads that are attached with a nucleic acid librarycan be deposited onto a surface or well in the presence of a polymercompound. In some embodiments, a polymer compound can inhibitelectro-osmotic flow to improve deposition of beads (e.g., attached withnucleic acids) onto a surface or well. A bead can be attached with anamplified (e.g., clonally amplified nucleic acid library). In someembodiments, a nucleic acid library bound to a bead can be depositedonto a surface or into a well in the presence of an aqueous solutioncomprising a polymer compound (e.g., a polyvinylpyrrolidone orderivative thereof).

In another example, a surface or well can be coated with a polymercompound. In some embodiments, a polymer compound can act as a blockingagent (e.g., a passivating agent) to reduce adherence of a surface tonucleic acids, beads or nucleic acid amplification reagents (e.g.,polymerase). A surface can include a surface of any component of amembrane-based emulsion-generating system (e.g., emulsion-generatingmembrane, gasket, wall of container), emulsion thermocycling subsystem(e.g., thermocycling plate, gasket, fluidics passageway) or a system forrecovering a polymerase chain reaction (e.g., fluidic distributiondevice, collection tube). For example, any surface of a thermocyclingplate (e.g., a fluid passageway) can be coated with a polymer compoundto reduce adherence between the surface and beads, nucleic acids, orreagents in a nucleic acid amplification reaction.

In some embodiments, microreactors can include at least one polymercompound (e.g., polyvinylpyrrolidone) at about 0.1-8%, or about 1-2%, orabout 2-3%, or about 3-4%, or about 4-5%, or about 5-6%, or about 6-7%,or about 7-8%.

In some embodiments, a microreactor comprises a plurality of templatebeads, a library of templates from a sample, DNA polymerase, a buffer,dNTPs, one or more surfactants, a pair of primers, and about 0.5-4%polymer compound, in an aqueous phase solution to form an aqueous phase.

In some embodiments, a microreactor comprises a plurality of templatebeads, a library of templates from a sample, DNA polymerase, a buffer,dNTPs, one or more surfactants, a pair of primers, and about 0.5-4%polyvinylpyrrolidone (e.g., 2% final concentration of PVP 10 kD or 40kD), in an aqueous phase solution to form an aqueous phase.

The aqueous phase can be contacted with an oil phase and emulsified toform an inverse emulsion comprising microreactors in an oil phase.

Once the inverse emulsion has been formed, DNA can be amplified withinthe individual microreactors by conducting emulsion polymerase chainreaction (ePCR) as shown in step 54 of FIG. 1. In some embodiments, theemulsion PCR step, step 54, can comprise thermally cycling the emulsionto cause respective polymerase chain reactions to occur within themicroreactors. The polymerase chain reactions can cause the formation ofa plurality of templated beads each comprising a plurality of amplicons,attached thereto, of the respective template originally contained in therespective microreactor. According to some embodiments, the polymerasechain reactions can produce, for example, more than 1,000 copies, morethan 10,000 copies, more than 30,000 copies, or more than 50,000 copiesof the template attached to each respective template bead.

Emulsion Generation Subsystem

To begin the ePCR, each template bead can comprise a respective primer,for example, a P1 primer, attached to a bead. In non-clonalmicroreactors, the template bead cannot amplify. Although beads arereferred to often herein, it is to be understood that other template ortarget supports can be used, for example, particles, granules, rods,spheres, shells, combinations thereof, and the like, which arecollectively referred to herein as “beads.” Furthermore, although themicroreactors are described herein as containing components for PCR, itis to be understood that the microreactors can contain components forreactions other than PCR, for example, components for an isothermalreaction, components for a different type of amplification reaction,components for an enzymatic reaction, components for a ligationreaction, or the like.

After emulsion PCR (ePCR) is complete, some of the template beadscomprise amplicons of the respective template, formed thereon. Suchbeads with amplicons are herein referred to as templated beads.Templated beads are template beads on which amplification hassuccessfully taken place in the respective microreactors. Some of thetemplate beads do not comprise amplicons of the template formed thereon,and are herein referred to as non-templated beads. Non-templated beadsare template beads on which no amplification took place in therespective microreactors. The non-templated beads can also be referredto as non-amplified or non-amplifying beads.

According to various embodiments, the templated beads can be collectedby breaking the emulsion to release the templated beads from themicroreactors. The collected template beads can then be washed. Breakingthe emulsion can comprise a chemical or physical treatment of theemulsion, for example, dispensing the emulsion in a centrifuge andcontacting the emulsion with a surfactant or other emulsion breakingagent. According to some embodiments, the emulsion can be contacted withan alcohol, for example, with propanol, butanol, pentanol, or the like.

In some embodiments, each of the templated beads and each of thenon-templated beads can have a diameter of from 0.1 μm to 5.0 μm, from0.25 μm to 2.0 μm, from 0.5 μm to 1.0 μm, from 0.7 μm to 1.2 μm, or from0.9 μm to 1.1 μm.

According to various embodiments, a wide range of different emulsionvolumes, for example, of from approximately 1 mL to 250 mL or more, canbe prepared without maintaining a stock of differently sized orconfigured consumables to accommodate a particular emulsion volume. Theemulsion can be made to exhibit a small drop size variation, a slow rateof reversion or phase separation, and an adaptability to a wide varietyof volume sizes.

In some embodiments, the present teachings provide devices, methods, andformulations for the preparation of inverse (water-in-oil) emulsions forpolymerase chain reactions. In various embodiments, a discrete aqueousphase (droplet) can entrap a particle, for example, a magnetic particleof about 1 μm diameter size and having a template oligonucleotideimmobilized on its surface. The discrete aqueous phase droplet can alsocomprise PCR reagents such as dNTPs, enzymes, co-enzymes, salts,buffers, surfactants, and a template molecule such as a DNA sample. Thetemplate molecule can be a sample DNA molecule, for example, a templatefrom a library of templates from a single sample. The carrier fluid cancomprise oil with or without added surfactants that havehydrophilic-lipophilic-balance (HLB) values of about 5.0 or less.According to various embodiments of the invention, the surfactants cancomprise a mixture of surfactants having various HLB values. Thoseskilled in the art can appreciate that the surfactant affinitydifference (SAD) of the oil phase can be adjusted by using varioussurfactants with various HLB values such that a stable inverse(water-in-oil) emulsion can be prepared.

In some embodiments, the carrier fluid can comprise a mineral oil suchas Petroleum Special, an alkane such as heptadecane, a halogenatedalkane such as bromohexadecane, an alkylarene, a halogenated alkyarene,a carbonate oil (e.g., Tegosoft DEC™), an ether, or an ester having aboiling temperature above 100° C., or any combination thereof. Thecarrier fluid can be insoluble or only slightly soluble in water. Theratio between the carrier fluid and the discrete aqueous phase canrange, for example, from 1/0.1 v/v to 4/1 v/v, from 0.5/1 to 3/1, from0.8/1 to 1/1, or as desired.

FIG. 2 depicts one embodiment of a machine or an automated samplepreparation system 62 for conducting automated sample preparation.Automated sample preparation system 62 can comprise a housing 66 forretaining an emulsion-generating station, an emulsion PCR station, and abead breaking/washing station, disposed within housing 66. Theemulsion-generating station can comprise a carrier fluid supply 70 and amicroreactor mixture supply 74 for use in the emulsion-generatingstation of the system. The emulsion PCR station can comprise a lever 78extending from a surface of housing 66 which can be lifted to an openposition whereby a PCR thermocycling plate can be inserted between afirst and a second heating block of the emulsion PCR station withinhousing 66. The bead breaking/washing station can comprise a centrifuge82 having a centrifuge cover 86 (shown closed), that is hingedlyattached to centrifuge 82. A display 64 can be provided to facilitatecontrol or monitoring of automated sample preparation system 62. Display64 can comprise a touch screen display, an LED display, a LCD screen, ora combination thereof. Display 64 can show the progress of the operationas it is carried out, and can display, for example, the percentcompletion of an operation while it is being carried out. According tovarious embodiments, with loading and one touch, a user can makeautomated sample preparation system 62 run without user interaction fora processing period, for example, for a period of one hour, two hours,three hours, or longer, to produce templated and amplified beads usefulfor sequencing.

Embodiments of the present teachings provide a filter basedemulsion-generating device. The filter can be any device having aplurality of pores of suitable sizes, including but not limited to amembrane and a filter plate. The emulsion-generating device may comprisea first channel plate, a second channel plate, and a first filterdisposed between the first channel plate and the second channel plate.The first filter may form a first chamber with the bottom surface of thefirst channel plate and a second chamber with the top surface of thesecond channel plate. In the present disclosure, a channel plate can bea gasket. The channel plate can be made of a hard material, includingbut not limited to glass, metal, and hard plastic. The channel plate canalso be made of a soft material, including but not limited to rubber andsoft plastic.

The first channel plate may comprise a first fluid port comprising anorifice passing through the first channel plate in a thicknessdirection. Thus, the first fluid port allows fluid to flow in or out ofthe first chamber. In an embodiment, the first fluid port is an inletfor introducing an aqueous reaction mixture, e.g., an aqueous ePCRmixture, into the emulsion-generating device.

The first channel plate may also comprise at least one first flowchannel disposed on its bottom surface, i.e., facing the first chamber.In some embodiments, the first channel plate may comprise two or morefirst flow channels disposed on its bottom surface. The first flowchannels may or may not contact or cross each other. In an embodiment,at least two of the first flow channels may form a contact or cross eachother. In another embodiment, the first flow channels do not formcontact or cross each other. In still another embodiment, the firstchannel plate may comprise a first flow channel that does not connect tothe first fluid port.

The second channel plate may comprise a second fluid port comprising anorifice passing through the second channel plate in a thicknessdirection. Thus, the second fluid port allows fluid to flow in or out ofthe second chamber. In an embodiment, the second fluid port is an inletfor introducing a carrier fluid, e.g., a water immiscible fluid, intothe emulsion-generating device.

The second channel plate may also comprise at least one second flowchannel disposed on its top surface, i.e., facing the second chamber. Insome embodiments, the second channel plate may comprise two or moresecond flow channels disposed on its top surface. The second flowchannels may or may not contact or cross each other. In an embodiment,at least two of the second flow channels may form a contact or crosseach other. In another embodiment, the second flow channels do not formcontact or cross each other. In still another embodiment, the secondchannel plate may comprise a second flow channel that connects to thesecond fluid port. In still another embodiment, the second channel platemay comprise at least one second flow channel that does not connect tothe second fluid port.

In another embodiment, the first or the second channel plate may furthercomprise a third fluid port comprising an orifice passing the secondchannel plate in a thickness direction. Thus, the third fluid portallows fluid to flow in or out of the first or the second chamber. In anembodiment, the third fluid port is an outlet for discharging theemulsion containing aqueous microreactor droplets in the carrier fluidfrom the emulsion-generating device. In an embodiment, the first or thesecond channel plate may comprise a flow channel that connects to thethird fluid port. In another embodiment, the first or the second channelplate may comprise a flow channel that does not connect to the thirdfluid port.

In still other embodiments, the emulsion-generating device of thepresent teachings may further comprise one or more second filtersdisposed between the first channel plate and the first filter, i.e., inthe first chamber, or one or more third filters disposed between thesecond channel plate and the first filter, i.e., in the second chamber.

In the present teachings, the filter, e.g., the first, second or thirdfilter, may comprise a plurality of pores, e.g., a plurality of poreshaving a size of about 1 to about 50 microns. In an embodiment, thefilter, e.g., the first, second or third filter, is a membrane. In aspecific embodiment, the filter is a track-etched filter. In the presentdisclosure, a pore of the filter is also referred to as a through hole.

The emulsion-generating device of the present teachings can furthercomprise a housing, in which the first channel plate, the second channelplate, and the first filter are mounted. Alternatively, the firstchannel plate and the second channel plate can also form the housing inwhich the first filter is mounted.

In some embodiment, the least one first flow channel or the at least onesecond flow channel is configured to have a low fluid resistance.

In some other embodiment, the at least one first flow channel or the atleast one second flow channel may comprise two ends connected by achannel, where the two ends and the channel are disposed in therespective first or second channel plate. In some other embodiment, theat least one first flow channel or the at least one second flow channelis disposed such that fluid passes said first filter a plurality oftimes.

In yet another embodiment, the first chamber or the second chamber has adepth from said first or second channel plate to said first filter of500 μm, and the at least one first flow channel or said at least onesecond flow channel has a depth of less than 500 μm.

The present teachings also provide a system for making emulsiondroplets. The system may comprise an emulsion-generating device of thepresent teachings, a reaction mixture supply in fluid communication withthe first chamber by way of said first fluid port; a carrier fluidsupply in fluid communication with the second chamber by way of saidsecond fluid port, and an emulsion collection device in fluidcommunication with the second chamber by way of the third fluid port.The emulsion collection device is any device to which the emulsion isoutput. For example, the emulsion collection device can be the emulsionPCR device 54. The system can also comprise a device for applying asuitable pressure in the reaction mixture supply or the carrier fluidsupply.

The present teachings also provide a method of making emulsion droplets,comprising: flowing an aqueous reaction mixture into the first fluidport of an emulsion-generating device of the teachings, flowing acarrier fluid immiscible with the aqueous reaction mixture into thesecond fluid port of the emulsion-generating device, and recovering anemulsion fluid comprising droplets of the aqueous reaction mixture inthe carrier fluid from the third fluid port. In an embodiment, themethod may further comprise adjusting a fluid pressure of the reactionmixture or adjusting a fluid pressure of the carrier fluid such that thedroplets of the reaction mixture in the carrier fluid in said step (d)have a predetermined size.

FIG. 3 depicts a schematic diagram and side view of a membrane-basedemulsion-generating system 90 for generating emulsion droplets,according to various embodiments. Emulsion-generating system 90 cancomprise an emulsion-generating device 94, a carrier fluid supply 98, amicroreactor droplet mixture supply 102, and an emulsion collectiondevice 106. Tubes 110, 114, and 118 can be provided to connectemulsion-generating device 94 to carrier fluid supply 98, microreactordroplet mixture supply 102, and emulsion collection device 106,respectively.

According to various embodiments, emulsion-generating device 94 cancomprise a top channel gasket 122, a bottom channel gasket 124, and anemulsion-generating membrane 126 disposed between top channel gasket 122and bottom channel gasket 124. According to various embodiments,emulsion-generating device 94 can be part of a machine where, withloading and one touch, a user can program the machine to run withoutfurther user interaction for a processing period, for example, for onehour, two hours, three hours, or longer, as is desired, to producetemplated beads useful for sequencing.

Top channel gasket 122, bottom channel gasket 124 andemulsion-generating membrane 126 can independently comprise a polymer,although other suitable, PCR-compatible materials can be used. Exemplarypolymers include poly(ether sulfone), polyester, polyisoprene,polycarbonate, polyvinylpyrrolidone, polyimide, polyamide,polytetrafluoroethylene (PTFE) or other fluorinated polymers orperfluorinated polymers, poly-cyclo-olefin polymers, silicone, and thelike. An exemplary silicone includes liquid silicone rubber (LSR).According to some embodiments, top channel gasket 122 and bottom channelgasket 124 can comprise rubber or polyisoprene. According to someembodiments, emulsion-generating membrane 126 can comprisepolyvinylpyrrolidone. Emulsion-generating membrane 126 can comprise ahydrophilic material or can be treated to exhibit hydrophilicproperties. In other embodiments, emulsion-generating membrane 126 cancomprise a hydrophobic material or can exhibit hydrophobic properties.The surface of emulsion-generating membrane 126 can be physically orchemically modified to tailor its hydrophilicity.

According to some embodiments, top channel gasket 122 and bottom channelgasket 124 can, themselves, form endwalls to support emulsion-generatingmembrane 126 there between. In other embodiments, a container 142 in theform of an outer container, housing, or enclosure, can be providedinside of which emulsion-generating membrane 126, top channel gasket122, and bottom channel gasket 124, can be disposed. Container 142 cancomprise a first portion and a second portion that can be glued, molded,ultrasonically bonded, screwed, or otherwise connected together to forma sealed housing around emulsion-generating membrane 126, top channelgasket 122, and bottom channel gasket 124. Shoulders, ridges, rims,protrusions, catches, or other features can be provided in the innersurfaces of container 142 to hold or maintain emulsion-generatingmembrane 126, top channel gasket 122, and bottom channel gasket 124 inplace. A microreactor input port 130 can be provided in a first portionor top portion 140 of container 142. A carrier fluid input port 134 andan emulsion output port 138 can be provided in a second portion orbottom portion 148 of container 142. Alternatively, an emulsion outputport 138 can be defined in top portion 140 of container 142.

It should be understood that carrier fluid supply 98 comprise apressurized supply of a carrier fluid. Similarly, microreactor dropletmixture supply 102 can comprise a pressurized supply of a microreactormixture. Pressure to the supplies can be independently provided by apump, for example, by a displacement pump, such as by a syringe pump, bya peristaltic pump, by a pneumatic pump, by an air pump, or the like.According to some embodiments, a control unit can be provided toalternate pressure provided to carrier fluid supply 98 and microreactordroplet mixture supply 102. Emulsion-generating system 90 can comprise afirst pressure source to provide an external pressure for moving themicroreactor stream through the plurality of through holes.Emulsion-generating system 90 can comprise a second pressure source toprovide an external pressure for moving the carrier fluid stream throughthe plurality of through holes. In some embodiments, the pressuresources can each comprise one or more syringe pumps.

All consumables in the system, including, device 94, container 142 andtubing 110, 114, and 118, can be disposable to minimizecross-contamination. Container 142 can comprise a polymer, such as apolyalkylene material, polyamide, silicone, fluoropolymer, or the like.Tubing 110, 114, and 118, can comprise a polymeric material, such as asilicone material, a polyalkylene material, a polyamide material, afluorpolymer, or the like.

As shown in FIG. 4A, according to some embodiments, container 142 caninclude positioning grooves 144 or rims or shoulders, so thatemulsion-generating membrane 126, top channel gasket 122, and bottomchannel gasket 124, are properly spaced apart from each other. Accordingto some embodiments, pins or other spacing elements can further bedisposed between emulsion-generating membrane 126 and top channel gasket122, and between emulsion-generating membrane 126 and bottom channelgasket 124, to further support, space, and position emulsion-generatingmembrane 126. According to some embodiments, emulsion-generatingmembrane 126 can be rigidly mounted in container 142, so that spacing ismaintained between emulsion-generating membrane 126, top channel gasket122, and bottom channel gasket 124. According to some embodiments, aspace between emulsion-generating membrane 126 and top gasket 122 candefine a first chamber 120. According to some embodiments, a spacebetween emulsion-generating membrane 126 and bottom gasket 124 candefine a second chamber 128.

According to various embodiments, top channel gasket 122 can comprise afirst gasket port 146 that can be aligned with microreactor input port130. Bottom channel gasket 124 can comprise a second gasket port 150that can be aligned with carrier fluid input port 134 and a third gasketport 154 that can be aligned with emulsion output port 138. According tosome embodiments, when emulsion output port 138 is defined in topportion 140 of container 142, third gasket port 154 can be defined intop channel gasket 122 instead of bottom channel gasket 124.

As shown in FIG. 4A, emulsion-generating membrane 126 can comprise aplurality of through holes 156. Each of the plurality of through holes156 can comprise a straight through pore that connects a first surfaceof emulsion-generating membrane 126 with a second opposing surface ofemulsion-generating membrane 126, and through which a straight line canbe drawn that does not touch or intersect the wall of the pore. Each ofthe plurality of through holes 156 can be circular in cross-section andthe plurality of them can be substantially uniform in size. According tosome embodiments, each of the plurality of through holes 156 can have adiameter of, for example, from about 1 to about 50 microns, from about 3to about 15 microns, from about 8 to about 14 microns, or from about 10to about 12 microns. According to some embodiments, a rim can extendfrom one or both surfaces of emulsion-generating membrane 126 aroundeach of through holes 156 although no rims are shown in the embodimentsdepicted. Emulsion-generating membrane 126 can be track-etched to formthe plurality of through holes 156. The track-etched through holes canbe fabricated using photo-lithography, chemical etching, reactive ionetching (RIE), or the like. According to some embodiments,emulsion-generating membrane 126 can be flexible. According to someembodiments, emulsion-generating membrane 126 can be rigid.

The emulsion generating membrane 126 can alternatively be formed by alaser etch process. For example, the laser etched holes formed in themembrane 126 can have a diameter from about 1 to about 50 microns, fromabout 3 to about 15 microns, from about 8 to about 14 microns, or fromabout 10 to about 12 microns. Alternatively, the laser etched holes canhave a diameter in a range of about 1 to 10 microns, such as a range of2 to 8 microns, a range of 3 to 7 microns, or approximately 5 microns.In a particular example, the membrane 126 can include laser etched holesthat correspond with the flow regions where channels are formed withinthe top channel gasket 122 and the bottom channel gasket 124. Inparticular, the laser etched membranes provide more uniform spacing andless variance in the diameter of through holes within the membrane.

FIG. 4B shows a top view of emulsion-generating device 94. Microreactorinput port 130 is shown provided in a top portion of container 142. Itshould be noted that container 142, shown in FIG. 4B, is transparentsuch that top channel gasket 122 can be seen through it.

FIG. 4C shows a bottom view of emulsion-generating device 94. Carrierfluid input port 134 and emulsion output port 138 are shown provided intransparent bottom portion 148 of container 142. It should be noted thatcontainer 142, shown in FIG. 4C is transparent such that bottom channelgasket 124 can be seen through it.

FIG. 4D shows an exploded view of the inner components ofemulsion-generating device 94. As depicted in FIG. 4D, top channelgasket 122 can comprise a first flow-path channel 170 and a secondflow-path channel 174, defined within the bottom surface of top channelgasket 122. First flow-path channel 170 and a second flow-path channel174 are shown only because top channel gasket 122 is transparent. Bottomchannel gasket 124 can comprise a third flow-path channel 178, a fourthflow-path channel 180, and a fifth flow-path channel 184 defined in thetop surface thereof. The flow-path channels of top channel gasket 122and bottom channel gasket 124 can be configured to have low resistance.In operation, the flows of a microreactor mixture stream 158, a carrierfluid stream 162, and a stream of emulsion droplets 166 can be directedby the flow-path channels. The flow-path channels can also direct andmanipulate the flows of partially formed emulsion streams 188, in whichemulsion droplets are formed but not necessarily of the proper size. Byflowing back and forth through emulsion-generating membrane 126 andalong the flow-path channels, the travel of the carrier fluid andmicroreactor mixture within emulsion-generating device 94 can bemaximized as can the opportunity for the emulsion components to be mixedtogether to form uniformly sized microreactor droplets encapsulated inthe carrier fluid. In this manner, the carrier fluid and microreactorcan be appropriately mixed to form uniformly sized emulsion dropletsbefore the emulsion is made to exit emulsion-generating device 94. Thirdflow-path channel 178, fourth flow-path channel 180, and fifth flow-pathchannel 184 can be situated such that the emulsion droplets can passthrough emulsion-generating membrane 126 two or more times before theresulting emulsion droplets are output in the form of a uniformemulsion. In some embodiments, the emulsion droplets can flow back andforth through emulsion-generating membrane for three, four, five, six,seven, eight, nine, or ten times, before exiting emulsion-generatingdevice 94, with many of the passage flow-throughs creating smaller andsmaller microreactor droplets.

FIG. 5 depicts another embodiment of an emulsion-generating device,generally designated as 190 in the drawings. Emulsion-generating device190 can comprise a top channel gasket 192 and a bottom channel gasket194. Emulsion-generating device 190 can be substantially similar toemulsion-generating device 94 shown in FIGS. 4A-4D with the exceptionthat a plurality of emulsion-generating membranes can be disposedbetween top channel gasket 192 and bottom channel gasket 194. A firstemulsion-generating membrane 196, a second emulsion-generating membrane198, and a third emulsion-generating membrane 200, can be disposedbetween top channel gasket 192 and bottom channel gasket 194, in astacked configuration with respect to one another. Top channel gasket192 and bottom channel gasket 194 can themselves form endwalls of acontainer or can be encased in an outer container, housing, orenclosure. Device 190 can comprise an outer container (not shown) insideof which emulsion-generating membrane 196, emulsion-generating membrane198, emulsion-generating membrane 200, top channel gasket 192, andbottom channel gasket 194, can be disposed. The outer container, notshown in FIG. 5, can be configured as described above with respect tocontainer 142. The outer container can comprise, for example, amicroreactor mixture supply input port, a carrier fluid input port, andan emulsion output port, as previously described.

Top channel gasket 192 can comprise a first gasket port 202 that can bealigned with the microreactor mixture supply input port of the outercontainer. Bottom channel gasket 194 can comprise a second gasket port204 that can be aligned with a carrier fluid input port of the outercontainer. A third gasket port 206 that can be aligned with an emulsionoutput port of the outer container. Emulsion-generating membranes 196,198, and 200 can comprise a plurality of through holes exemplified as208, 210, and 212, respectively. The plurality of through holes in theemulsion-generating membranes can each be circular in cross-section andthe plurality of them can be substantially uniform in size. Top channelgasket 192 can comprise a first flow-path channel 214 and a secondflow-path channel 216 defined within a bottom surface of top channelgasket 192 and visible in FIG. 5 only because top channel gasket 192 istransparent. Bottom channel gasket 194 can comprise a third flow-pathchannel 218, a fourth flow-path channel 220, and a fifth flow-pathchannel 222 defined within a top surface of bottom channel gasket 194. Amicroreactor mixture stream 224, a carrier fluid stream 226, and astream of emulsion droplets 228 can travel through the through holes ofthe emulsion-generating membranes and can be directed and manipulated bythe flow-path channels toward and through third gasket port 206.

The channel gaskets 122 and 124 provide flow paths through the membrane126 so that the emulsion traverses the membrane 126 more than once suchas at least 2 times, at least 3 times or even more. In the embodimentsillustrated in FIGS. 4D and 5, the channels 170, 174, 178, 180, and 184or 214, 216, 218, 220, and 222 form complementary passageways in whichthe carrier fluid and emulsion pass through the membrane more than once.In the illustrated embodiment of FIG. 4D, the complementary flow path iscountercurrent. For example, when the emulsion passes from the bottomchannel gasket 124 through the membrane 126 to the top channel gasket122, the fluid traveling within the channel of the bottom channel gasket124 is flowing in an opposite direction as a fluid passing through thechannel of the top channel gasket 122. Such flow is referred to ascountercurrent flow providing a countercurrent complementary flow pathbetween the gaskets 122 and 124.

In an example illustrated in FIG. 32, a channel 1602 of a channel gasket1600 flow in one direction along portions of the channel 1602 thatoverlap with the channel 1604 of the top channel gasket. The channel1604 is illustrated in broken lines indicating its disposition on adifferent channel gasket. Accordingly, the carrier fluid enters throughport 1608 traversing back-and-forth across the membrane through acountercurrent complementary flow path and an emulsion exits throughport 1606.

In an alternative example, FIG. 33 illustrates the flow path 1700 thatincludes concurrent flow. For example, flow of a carrier fluid can enterport 1708 and traverse channels 1702. In regions of overlap, the flowdirection in the channel 1702 is the same as the flow direction inchannel 1704 of the opposing gasket. As such, the flow is concurrent orparallel in the overlapping regions as emulsion passes from the bottomgasket through the membrane to the top gasket. Similarly, the flow isconcurrent when the emulsion passes from the top gasket through to thebottom gasket. As such, FIG. 33 illustrates an exemplary concurrentcomplementary flow path 1700.According to various embodiments, a methodof making a membrane-based emulsion can comprise pumping or dispensing avolume of a microreactor mixture in the microreactor input port of anemulsion-generating membrane device to form a microreactor mixturestream. Simultaneously or sequentially, a volume of carrier fluid can bepumped or dispensed through a carrier fluid input port of theemulsion-generating membrane device to form a carrier fluid stream.External pressure can be applied to move the microreactor stream and thecarrier fluid stream, for example, alternatively, through the throughholes of the emulsion-generating membrane to cause the carrier fluidstream and the microreactor stream to mix together and form an emulsionstream comprising microreactor droplets.

According to some embodiments, the emulsion stream of microreactordroplets can be directed by the flow-path channels of the top channelgasket and of the bottom channel gasket to the through holes of theemulsion-generating membrane or to the third gasket port. The stream ofemulsion droplets can travel through the through holes of theemulsion-generating membrane one or more times before exiting the devicethrough the output port. The emulsion droplets can be sheared intosmaller droplets as the emulsion droplets are pushed back and forththrough the through holes. The emulsion droplets can be pushed back andforth through the through holes, two or more times, for example, six,seven, eight, nine, or ten times, such that microreactor droplets ofuniform size and volume are generated.

In an embodiment, the present teachings provide a plate-basedemulsion-generating device. The emulsion-generating device may comprisea first plate and a second plate configured to form a flow chamber; afluid input port and a fluid output port in fluid communication with theflow chamber; at least the first or second plate comprising a pluralityof through holes passing through the first or second plate in a platethickness direction; the plurality of through holes being disposed inone or more lines oriented at a substantially perpendicular directionwith respect to a direction from the fluid input port toward the fluidoutlet port. In an embodiment, the plurality of through holes isdisposed in one or more straight lines. However, none straight lines,e.g., curves and zigzag lines are also contemplated. A person skilled inthe art would readily select a suitable arrangement of the through holesas long as, inter alia, the droplets generated from different throughholes would not interfere with each other. In an embodiment, the devicecan further comprise a respective elevated rim around each of thethrough hole.

In another embodiment, the first plate is a top plate and the secondplate is a bottom plate, and the bottom plate may comprise the pluralityof through holes.

In another embodiment, the present teachings provide a system for makingemulsion droplets, comprising a plate-based emulsion-generating device,a reaction mixture supply in fluid communication with the flow chamberby way of the plurality of through holes, a carrier fluid supply influid communication with the flow chamber by way of the fluid inletport, and an emulsion collection device in fluid communication with theflow chamber by way of the outlet fluid port. In an embodiment, thereaction mixture supply or the carrier fluid supply are pressurized.

The present teachings also provide a method of making an inverseemulsion from a reaction mixture and an immiscible carrier fluid usingthe plate-based emulsion-generating device. The method may compriseflowing the reaction mixture into the flow chamber through the throughholes, flowing the carrier fluid into the flow chamber through the fluidinlet, and forming a plurality of droplets of the reaction mixture inthe carrier fluid, thus forming an inverse emulsion. The method canfurther comprise recovering the inverse emulsion from the fluid outletport. In an embodiment, the method may comprise adjusting a fluidpressure of the reaction mixture or adjusting a fluid pressure of thecarrier fluid such that the droplets of the reaction mixture in thecarrier fluid have a predetermined size. In a preferred embodiment, thereaction mixture may comprise an aqueous phase solution, a plurality oftemplate beads, a library of templates from a sample, DNA polymerase,and a pair of primers.

In an alternative embodiment, an aqueous mixture is placed in a samplevial, which is coupled to the top port of an emulsion generating device.A displacement fluid rises into the sample vial pushing the more denseaqueous solution into the emulsion generating device. For example, FIG.25, FIG. 26, FIG. 27, FIG. 28, FIG. 29, FIG. 30, and FIG. 31 illustratealternative embodiments of the emulsion generating device. For example,an emulsion generating device 1500 has upper portion 1502 and a lowerportion 1504. The emulsion generating device 1500 also includes a topchannel gasket 1506, and bottom channel gasket 1510, and an emulsiongenerating membrane 1508 disposed between the top channel gasket 1506and the bottom channel gasket 1510. In an example, the membrane 1508 canbe separated from the top channel gasket 1506 or the bottom channelgasket 1510 by a distance of at least 500 μm. In an alternative example,the membrane 1508 is in direct contact with the top channel gasket 1506or the bottom channel gasket 1510, restricting fluid flow to the channelregions within the gaskets 1506 or 1510. At the top portion 1502 of theemulsion generating device 1500, the port 1514 is to receive a samplevial 1512, providing fluid access between the sample vial 1512 andemulsion generating device 1500.

The bottom portion 1504 of the emulsion generating device 1500 caninclude carrier fluid input port 1516 for inserting carrier fluid, suchas oil. On the bottom portion 1504, the emulsion generating device canalso include an emulsion outlet port 1518 to receive the generatedemulsion. Such carrier fluid input port 1516 and the emulsion outletport 1518 can function as described above.

In an additional example, the bottom portion 1504 of the emulsiongenerating device 1500 can include a displacement fluid input port 1520.In an example, a passageway 1530 can be formed within the top channelgasket 1506, the membrane 1508, and the bottom channel gasket 1510 topermit the flow of the displacement fluid from the displacement fluidport 1520 to the sample vial 1512 through the sample port 1514. In anexample, the displacement fluid applied through the displacement port1520 can be a fluid having similar properties to the carrier fluid.Alternatively, displacement fluid can be different than the carrierfluid. Optionally, the displacement fluid is miscible in the carrierfluid. Alternatively, the displacement fluid is immiscible in a carrierfluid. In a particular example, the displacement fluid is immisciblewith the aqueous solution and has a density less than the aqueoussolution. While the displacement fluid input port 1520 is illustrated asbeing part of the bottom portion 1504 of the emulsion generating device1500, alternatively, the displacement fluid input port 1520 can beformed in the upper portion 1502 of the emulsion generating device 1500.Alternatively, the system can be free of a displacement fluid input port1520 and the fluid path of the carrier fluid to be fed through thecarrier fluid input port 1516 can be directed to displace the aqueoussolution within the sample vial 1512.

In a further example illustrated in FIG. 36 and FIG. 37, displacementfluid is introduced into a sample vial through a snorkel device. Asillustrated in FIG. 36, an emulsion generating device 1902 is attachedto a sample vial 1904, which defines a chamber 1906 for receiving asample solution and displacement fluid. A snorkel device 1908 forms aportion of a fluid pathway 1910 extending from a displacement fluidinlet port 1912 formed within a lower portion 1916 of the emulsiongenerating device 1902. The pathway 1910 extends through a lower channelgasket 1918, an emulsifying membrane 1920, and an upper channel gasket1922. The upper portion 1924 of the emulsion generating device 1902 cansecure the snorkel 1908 to further define the pathway 1910.

The lower portion 1916 of the emulsion generating device 1902 operatesas described above and includes the emulsion outlet port 1914 and acarrier fluid inlet port (not illustrated). In an example, carrier fluidis introduced by carrier fluid inlet port (not illustrated), whichcarries fluid and the sample through a tortuous path between a lowerchannel gasket 1918 and an upper channel gasket 1922 through anemulsifying membrane 1920. An emulsion resulting from such an operationexits the emulsion outlet port 1914.

Displacement fluid, such as an immiscible fluid having a lower densitythan the aqueous sample solution, can be supplied through thedisplacement fluid port 1912 through the displacement fluid pathway 1910and the snorkel device 1908 to the chamber 1906. The displacement fluid,being immiscible and less dense than the aqueous sample solution, floatson top of the sample solution and drives the sample solution through achannel 1926 into the tortuous path defined by the lower channel gasket1918, the upper channel gasket 1922, and the membrane 1920 positionedbetween the upper channel gasket 1922 and the lower channel gasket 1918.When the operation is complete, the top of the snorkel device 1908 issurrounded by displacement fluid within the chamber 1906, thuspreventing backflow of any remaining sample solution. In particular, itis been found that such a snorkel device 1908 prevents contaminationfrom reaching inlets to the device, thus reducing contamination duringfuture use of the device.

To prepare such an apparatus, the sample can be loaded into the vial1904 when the vial 1904 is detached from emulsion generating device1902. Alternatively, the sample can be fed through the displacementfluid inlet port 1912 through the pathway 1910 and the snorkel device1908 into the chamber 1906. In a particular example, the device may beheld upside down so that the vial 1914 is positioned below the emulsiongenerating device 1902. Following sample loading, an initial amount ofdisplacement fluid can be introduced through the port 1912 and along thepath 1910 through snorkel 1908 and into the chamber 1906 of the vial1904.

Once sufficient displacement fluid has been provided, the device can beturned over and applied to a PCR system, for example, in the orientationillustrated in FIG. 36. In a particular example, when turning over thedevice, positioning vial 1904 over the emulsion generating device 1902,the turning motion of the device can be enacted to facilitate movementof the displacement fluid to remain in contact with the upper opening ofthe snorkel 1908 to prevent contact between the opening of the snorkel1908 and the aqueous sample solution. Such a method of installing anemulsion generator may prevent further backflow contamination into theinlet of the system. In particular, the prevention of such contaminationin the inlet of the system can reduce errors caused, for example, byprimer dimerization and other contamination.

Yet another embodiment of an emulsion-generating device and subsystem isshown in FIGS. 21A-23. According to some embodiments, other devices andsubsystems can be used to generate emulsions. In some embodiments, aflow cell or plate-based emulsion-generating device can be provided toform emulsion droplets comprising microreactors. In the embodiment shownin FIG. 21A, an emulsion-generating device 1400 is shown comprising asubstrate 1404 at least partially defined by an emulsion-generatingplate 1408 and a cover 1412 disposed over and in contact with theperiphery of emulsion-generating plate 1408. Emulsion-generating plate1408 can comprise at least one row 1416 of one or more nozzles orthrough holes 1420 formed through the plate. According to variousembodiments, emulsion-generating plate 1408 can comprise at least twothrough holes 1420 defined through the plate. A flow cell 1424 isprovided within substrate 1404 and defined as the volume betweenemulsion-generating plate 1408 and cover 1412. The at least one row 1416of one or more through holes 1420 can be oriented in a straight linewithin flow cell 1424. Row 1416 can be arranged perpendicularly orangled within about 10 degrees or less with respect to perpendicular,relative to a direction of flow of a carrier fluid and resultingemulsion through the flow cell. The direction can be a direction from aflow cell input toward a flow cell output. A flow cell ledge or wall1428 can be disposed around the perimeter of flow cell 1424.

According to various embodiments, cover 1412 can comprise a carrierfluid inlet port 1432 through which a carrier fluid 1434 can enter flowcell 1424. The arrows representing flow 1434 are schematic in nature andshown in the interior of flow cell 1424 by virtue of cover 1412 beingtransparent. Alternatively, cover 1412 can be opaque, non-transparent,translucent, black, non-light-transmissive, or the like. According tovarious embodiments, each through hole 1420 can comprise a microreactormixture inlet port. Carrier fluid inlet port 1432can be circular incross-section and can be the same size, smaller than, or larger than anemulsion outlet port 1436 also formed in cover 1412. A flow of carrierfluid can be made through flow cell 1424 by supplying a carrier fluidunder pressure through carrier fluid inlet port 1432 to fill flow cell1424 and exit emulsion outlet port 1436. A volume of microreactormixture can then be dispensed through a through hole 1420 and into theinterior of flow cell 1424. The microreactor mixture can be forcedthrough emulsion-generating plate 1408 through each of the through holes1420 in row 1416 and into the flow of carrier fluid through flow cell1424. Emulsion droplets 1440 can be formed once the microreactor mixtureis forced out of emulsion-generating plate 1408 through a through hole1420 and sheared off of the microreactor supply remaining in the throughhole. The flow of the carrier fluid, the microreactor mixture, or bothcan be adjusted to control the shearing effect and the size of themicroreactor droplets sheared from the through holes. Emulsion outletport 1436, through which the resultant inverse emulsion 1440 leaves theemulsion-generating device, can be in fluid communication with acollection device, for example, a collection tube. In some embodiments,emulsion outlet port 1436 can be in direct or indirect fluidcommunication with a thermocycling plate, for example, a thermocyclingplate as described herein. The walls of flow cell 1424 can retaincarrier fluid 1434 and inverse emulsion 1440 within flow cell 1424.

Emulsion-generating plate 1408 and cover 1412 can be glued, molded,ultrasonically bonded, screwed, or otherwise connected together to formsubstrate 1404. According to various embodiments, emulsion-generatingdevice 1400 can be part of a machine wherein, with loading and onetouch, a user can program the machine to run without user further userinteraction for a processing period, for example, one hour, two hours,three hours, or longer, to produce amplified templated beads useful forsequencing.

FIG. 21B is a cross-sectional view taken along line B-B of FIG. 21A. Asshown in FIG. 21B, each of through holes 1420 can be a straight throughpore through which a straight line can be drawn that does not touch orintersect the wall of the pore. Each of the through holes 1420 can becircular in cross-section and can be substantially uniform in size.According to some embodiments, each of the through holes 1420 can have adiameter of, for example, from about 1 to about 30 microns, from about 3to about 15 microns, from about 8 to about 14 microns, or from about 10to about 12 microns. According to some embodiments, flow cell 1424 cancomprise a row of about 50 to about 100 through holes. Through holes1420 can be arranged substantially perpendicular to the flow of carrierfluid through flow cell 1424. According to some embodiments, flow cell1424 can comprise from about 60 to about 90 through holes. According tosome embodiments, flow cell 1424 can comprise from about 70 to about 80through holes. According to some embodiments, flow cell 1424 cancomprise about 60, about 70, about 80, about 90, or about 100 throughholes.

According to various embodiments, a respective rim or nozzle 1444 can beformed or provided on emulsion-generating plate 1408 around each of theone or more through holes 1420. Nozzle 1444 can help to make droplets ofuniform size or to eject the droplets at a location in flow cell 1424that is spaced from the flow cell floor. Nozzle 1444 can reduce oreliminate the possibility of droplets merging with each other. In someembodiments, nozzle 1444 is not surface treated, or does not comprise asurface coating. According to some embodiments, nozzle 1444 can betreated or can be provided with a surface coating. In some embodiments,nozzle 1444 can comprise a semiconductor-type material, for example,silicon.

As shown in FIG. 22, emulsion-generating plate 1408 can also contain,define, or at least partially define, a cavity 1448 that, in operation,is filled with the microreactor mixture and in fluid communication withthrough holes 1420. As shown, the top surface of emulsion-generatingplate 1408 can face cover 1412. Flow cell 1424 can be in the form of awell or chamber at least partially defined by the top surface ofemulsion-generating plate 1408. Cavity 1448 can be in the form of aninterior cavity inside emulsion-generating plate 1408 or it can definedby both emulsion-generating plate 1408 and a bottom plate or cover (notshown). In some embodiments, cavity 1448 can be drilled from a side ofemulsion-generating plate 1408 and sealed on the end by a side wall (notshown). Through holes 1420 can provide fluid communications between flowcell 1424 and cavity 1448. In some embodiments, cavity 1448 andemulsion-generating plate 1408 can be configured to sit directly on, orconnect to, a supply source, port, or connection for a microreactormixture supply.

According to various embodiments, a pressurized supply of a microreactormixture and a pressurized supply of carrier fluid can be connected toemulsion-generating device 1400. The pressurized supply of microreactormixture can be connected to a microreactor inlet port 1430. Thepressurized supply of carrier fluid can be connected to a carrier fluidinlet port 1432. In the embodiment shown, the microreactor mixture fromthe pressurized supply of microreactor mixture can be dispensed intoemulsion-generating plate 1408 through inlet port 1430 and through amicroreactor mixture conduit 1438 in fluid communication with inlet port1430. The microreactor mixture can travel downwardly intoemulsion-generating plate 1408, in a direction away from the top surfaceof emulsion-generating plate 1408, then horizontally through conduit1438 in a direction parallel to the top surface of emulsion-generatingplate 1408. The flow of microreactor mixture, denoted as referencenumeral 1452, can then travel upwardly through cavity 1448 and throughholes 1420. At about the same time that microreactor mixture isdispensed into emulsion-generating plate 1408, the carrier fluid fromthe pressurized supply of carrier fluid can be dispensed into flow cell1424 through carrier fluid inlet port 1432. The carrier fluid can flowin flow cell 1424 in a direction that is parallel to the top surface ofemulsion-generating plate 1408 and toward emulsion outlet port 1436. Themicroreactor mixture can be forced out of emulsion-generating plate 1408through the through holes 1420 and into the carrier fluid flowingthrough flow cell 1424. As the carrier fluid flow shears portions of themicroreactor mixture from nozzles 1444, microreactor droplets 1440 areformed.

According to some embodiments, as shown in FIG. 23, each nozzle 1444 canhave a small, elevated, top surface 1456 to help minimizesurface-wetting-induced size variability. According to some embodiments,top surface 1456 can be substantially flat. According to someembodiments, top surface 1456 can have a uniform width of about 2microns to about 11 microns in a radial direction. In some embodiments,nozzle 1444 can have an inside diameter of from about 1 micron to about40 microns, or from about 10 microns to about 20 microns. According tosome embodiments, the one or more through holes 1420 can each have aninside diameter of from about 1 to about 40 microns or from about 11 toabout 18 microns. Each nozzle 1444 can extend from about 10 microns toabout 20 microns above the flow cell bottom. According to someembodiments, nozzle 1444 can extend about 15 microns above the flow cellbottom. In some embodiments, nozzle 1444 can have an inside diameter ofabout 11, 12, 13, 14, or 15 microns. In some embodiments, nozzle 1444can have an outside diameter of from about 20 microns to about 30microns. According to some embodiments, nozzle 1444 can have an outsidediameter of about 22, 23, 24, 25, 26, or 27 microns.

Each nozzle 1444 can have any appropriate shape. Each nozzle 1444 can becylindrically-shaped, tear-drop shaped, square-shaped, or the like. Theplurality of nozzles can be spaced from one another at a pitch of fromabout 10 to about 50 microns, for example, from about 30 to about 40microns. In some embodiments, the plurality of through holes 1420 can bespaced apart at a pitch of about 33, 34, 35, 36, or 37 microns.

Emulsion-generating plate 1408 can be molded, machined, drilled, ortrack-etched to form the one or more through holes. Track-etched throughholes can be fabricated using photo-lithography, chemical etching, andreactive ion etching (RIE). Emulsion-generating plate 1408 can be rigidor flexible and can comprise plastic, a polymer, glass, metal, silicon,a combination thereof, or the like. In some embodiments,emulsion-generating plate 1408 can comprise a glass or a polymer.Exemplary polymers that can be used include, but are not limited to,poly(ether sulfone), polyester, polyisoprene, polycarbonate,polyvinylpyrrolidone, polyimide, polytetrafluoroethylene (PTFE),fluorinated polymers, perfluorinated polymers, poly cyclo-olefins,combinations and blends thereof, and the like.

Emulsion PCR and Thermocycling Subsystem

An emulsion generated can be thermally cycled, or thermocycled, toeffect a polymerase chain reaction in each aqueous droplet. For suchpurpose, a thermocycling subsystem is provided according to the presentteachings and can comprise a first thermocycling plate, as describedherein, and a heating subassembly, as described herein. The heatingsubassembly of the thermocycling system can also comprise acomplementary heating block as described herein. The heating subassemblyof the thermocycling system can also comprise a second thermocyclingplate.

A heating subassembly is provided by the present teachings that can beused in combination with one or more thermocycling plates describedherein. The heating subassembly can comprise a first heating block, afirst heat control unit, a second heat control unit, and, optionally, anegative load device. The first heating block can comprise a firststatic heating zone, a heating zone partition, and a second staticheating zone separated from the first static heating zone by the heatingzone partition. The first heat control unit is operably associable withthe first static heating zone. The second heat control unit is operablyassociable with the second static heating zone. The negative load devicecan be operably associated with the second heat control unit and thesecond static heating zone. Any type of negative load device can be usedthat can exert a negative temperature effect. For example, the negativeload device can comprise a fan, a heat sink, or both. A power source canbe electrically associated with the first and second heat control units.

The shape of the one or more heating blocks employed can adapted forcompatibility with the one or more thermocycling plates used. Forexample, one or more heating blocks of the heating subassembly cancomprise one or more recess configured to allow passage of athermocycling plate inlet, a thermocycling plate outlet, or tubingassociated with at least one of the inlet and the outlet, or anycombination thereof. Any type of heater or combination of heaters canemployed for the static heating zones. Examples of heaters includenarrow strip (ribbon) heaters, fluid-filled channel heaters, Peltierheaters, static zone heaters, electrical resistance heaters, and thelike.

FIG. 6A is a thermocycling subsystem of an embodiment of the presentteachings. thermocycling subsystem 300 is shown with four components ina sandwiched configuration. A first thermocycling plate 304 and a secondthermocycling plate 308 are shown sandwiched between a first heatingblock 312 and a second heating block 316. FIG. 6B shows an exploded viewof the thermocycling subsystem 300 as shown in FIG. 6A. FIG. 6Bhighlights and shows the portions of the respective heating blocks thatcorrespond to the respective thermocycling plates. More details of thesevarious components are described below in conjunction with the otherfigures. FIG. 6C shows another thermocycling subsystem 320, which is avariation on the thermocycling subsystem 300. Thermocycling subsystem320 again shows heating blocks 312 and 316, but with only a singlethermocycling plate, here second thermocycling plate 308. Otherembodiments consistent with the present teachings consisting ofvariations on the thermocycling subsystems 300 and 320 will be readilyunderstandable to one of ordinary skill in the art. For example, avariation on thermocycling subsystem 320 could instead involve firstthermocycling plate 304 instead of second thermocycling plate 308. Asingle heating block can be employed in those embodiments where a singlethermocycling plate is present. FIG. 6D shows an exploded view ofthermocycling subsystem 320 as shown in FIG. 6C analogous to theexploded view of FIG. 6A shown in FIG. 6B.

FIG. 7A shows a heating subassembly 322 and FIG. 7B shows a secondheating subassembly 350, which is a mirror image of subassembly 322.Subassembly 322 can comprise first heating block 312. First heatingblock 312 comprises a first static heating zone 324, a second staticheating zone 328, and an insulation/buffer zone 332 between first staticheating zone 324 and second static heating zone 328. First staticheating zone 324 can be operably connected to and controlled by a firstheat control unit 336, and second static heating zone 328 can beoperably connected to second heat control unit 340 and controlled by thesame. Heating subassembly 322 can also include a negative load device344, which can be operably connected to and controlled by second heatcontrol unit 340.

Both the first heat control unit 336 and the second heat control unit340 can be operably connected to a first power source 348. Any type ofpower source, whether alternating current, direct current, or othertype, can be used as the power source 348. Negative load 344 can be anydevice or means known to one of ordinary skill in the art for applying anegative temperature load. For example, negative load 344 can be in theform of a fan. The first static heating zone 324 and second staticheating zone 328 can extend through any degree of the thickness of theheating block 312. First heating block 312 can have an access recess 380in a corner to allow access of tubing connected to the firstthermocycling plate 304.

While a single heating block can be employed in the heating subassembly,use of a second heating block can provide additional temperature controland is particularly advantageous when two thermocycling plates are used.Accordingly, the heating subassembly of the present teachings cancomprise a complementary heating block. The complementary heating blockcan include a first complementary static heating zone, a complementaryheating zone partition, and a second complementary static heating zoneseparated from the first complementary static heating zone by thecomplementary heating zone partition. The complementary heating blockcan share one or more heat control units, or other components, with thefirst heating block or can be provided with dedicated complementary heatcontrol units or other components. For example, the complementaryheating block can comprise a first complementary heat control unitoperably associated with the first complementary static heating zone; asecond complementary heat control unit operably associated with thesecond complementary static heating zone; and a complementary negativeload device operably associated with the second complementary heatcontrol unit and the second complementary static heating zone. Thesecond heating block can be electrically associated with the firstcomplementary and second complementary heat control units.

Second (complementary) heating subassembly 350 shown n FIG. 7B can beconfigured to have same or similar attributes as described for firstheating subassembly 322. Second heating subassembly 350 includes secondheating block 316. Second heating block 316 comprises first staticheating zone 352, second static heating zone 356, and insulation/bufferzone 360. First heat control unit 364 and second heat control unit 368are respectively operably associable with and can control first staticheating zone 352 and second static heating zone 356. Negative loaddevice 372 is operably connected to and can be controlled by second heatcontrol unit 368. While first heat control unit 364 and second heatcontrol unit 368 are shown operably connected to second power source376, first heat control unit 364 and second heat control unit 368 canshare a common power source 348 with first heating subassembly 322.Second heating block 316 is also shown with an access/recess 384analogous to access/recess 380 in the first heating block 312.

The heat control units can be programmed or configured to maintain orvary the temperature or temperature range of the static heating zones.For example, the first heat control unit is configured to maintain thefirst static heating zone at a first temperature or within in a firsttemperature range. The second heat control unit can be configured tomaintain the first static heating zone at a second temperature or withinin a second temperature range. The first temperature can be higher thanthe second temperature, or vice versa. The first temperature range canbe higher than second temperature range, or vice versa. The firsttemperature range can be configured so as not to overlap with the secondtemperature range. The first temperature or temperature range, or thesecond temperature or temperature range, can be less than 1° C., fromabout 1° C. to about 1,000° C., from about 5° C. to about 500° C., fromabout 10° C. to about 150° C., from about 25° C. to about 125° C., fromabout 40° C. to about 115° C., from about 45° C. to about 75° C. fromabout 50° C. to about 105° C., from about 55° C. to about 100° C., fromabout 60° C. to about 98° C., from about 65° C. to about 95° C., fromabout 70° C. to about 94° C., from about 75° C. to about 80° C. to about92° C., from about 85° C. to about 100° C., from about 88 to about 94°C., or greater than 1,000° C. The first temperature or temperature rangecan differ from the second temperature or temperature range by less thanabout 5° C., from about 5° C. to about 50° C., from about 10° C. toabout 30° C., from about 15° C. to about 25° C., or more than about 50°C. The first temperature or temperature range, or the second temperatureor temperature range, can be a temperature or temperature rangesufficient to allow for denaturing of double-stranded nucleic acid,annealing of nucleic acids, or extension of nucleic acids, or anycombination thereof.

Accordingly, first heat control units 336 and 364, along with theirrespective first static heating zones 324 and 352, are generallyadaptable and configured to supply a temperature or temperature range orranges capable of denaturing double-stranded nucleic acid such as DNA.The denaturing temperature range can be from about 80° C. to about 120°C., from about 85° C. to about 115° C., from about 90° C. to about 105°C., from about 92° C. to about 100° C., or about 94° C. Second heatcontrol units 340 and 368 are adapted and configured to control theirrespective second static heating zones 328 and 356 to a temperature ortemperature range or temperature ranges serving to anneal and allow forextension of single-stranded nucleic acid. The temperature setting forthe second static heating zones 328 and 356 can be from about 40° C. toabout 80° C., from about 50° C. to about 75° C., from about 55° C. toabout 70° C., from about 58° C. to about 64° C. or about 62° C. Negativeloads 344 and 372 are configurable to supply a negative temperatureforce to their respective static heating zones 328 and 356 to force atemperature or temperature range below that set for the respectivestatic heating zones. Second heat control units 340 and 368 thenorchestrate a temperature heat application through the respective secondstatic heating zones to achieve a set temperature or temperature rangefor the respective second static heating zones.

A thermocycling plate is provided in accordance with the presentteachings, which can be used in conjunction with the heating subassemblyin a thermocycling subsystem. The thermocycling plate is particularlyadvantageous for performing polymerase chain reactions (PCR). Thethermocycling plate can generally comprise a slab housing and a mainfluid passage passageway that passes through the slab housing. The slabhousing can be configured to have any suitable geometry. For example,the slab housing can have a width, a length, and a thickness less thanboth the width and the length. The slab housing can have a plurality ofcorners comprising a first corner, a second corner, a third corner, anda fourth corner, as well as a plurality of edges comprising a first edgeextending from the first corner to the second corner, a second edgeextending from the second corner to the third corner, a third edgeextending from the third corner to the fourth corner, and a fourth edgeextending from the fourth corner to the first corner. A plurality ofpartitions can be provided in slab housing. For example, the partitionscan extend across the width and comprise a first partition proximal thefirst edge, a second partition between the first partition and a thirdpartition, and the third partition proximal the third edge. Thethermocycling plate can further comprise a first face bounded by theplurality of edges and plurality of corners; and a second face parallelto the first face; bounded by the plurality of edges and plurality ofcorners. The inlet, the outlet, or both can be located on the first faceor the second face. The inlet, the outlet, or both can be located at thefirst corner, the second corner, the third corner, or fourth corner. Theinlet, the outlet, or both can be located along the first edge, thesecond edge, the third edge, or the fourth edge.

The main fluid passageway is disposed in the slab housing and caninclude an inlet, an outlet, and various fluid passage segments in fluidcommunication with adjoining fluid passages. For example, the main fluidpassageway can be constructed to provide an inlet proximal a firstcorner. An initial fluid passage in fluid communication with the inletcan extend to proximal the second corner along the width of the slabhousing, between the first partition and the second partition. Atransition fluid passage in fluid communication with the initial fluidpassage can extend from proximal the second corner to proximal the thirdcorner along the length of the slab housing. A main fluid passage influid communication with the transition fluid passage can extend fromproximal the third corner to proximal the fourth corner along the widthof the slab housing, and between the second partition and the thirdpartition. The main fluid passage, the initial fluid passage, or bothcan have a tortuous shape. The shape can be winding, twisting, curvy,circuitous, serpentine, zigzag, meandering, crooked, labyrinthine,undulating, twisted, tangled, interweaved, or convoluted. The main fluidpassage, the initial fluid passage, or both can have a plurality ofcycles between their respective partitions. An outlet is provided influid communication with the main fluid passage. The inlet or outlet canbe configured to have any suitable form. For example, the inlet oroutlet can comprise an extension adapted to accept plastic tubing. Theplastic tubing can be wholly external to the thermocycling device, passthrough the fluid passageway partially, or completely pass though thefluid passageway. The fluid passageway can comprise an exit fluidpassage in fluid communication with the main fluid passage and extendingfrom proximal the fourth corner to proximal the first corner along thelength of the slab housing, with the outlet is proximal the first cornerin fluid communication with the exit fluid passage. In an embodiment,the main fluid passage, the initial fluid passage, or both are disposedat the first face of the sample reaction plate.

The spacing and arrangement of the transition and main fluid passage inthe thermocycling plate can be defined, in part, by partitions or areasgenerally devoid of fluid passageways. The second partition can beparallel to, or displaced at an oblique angle relative to, at least oneof the first partition and the third partition. Between variousembodiments, the distance between the first and second, second andthird, or first and third partitions can be varied. For example, thedistance between the second and third partitions can be greater than thedistance between the first and second partitions. Such a configurationallows for the main fluid passage to encompass a greater area, andvolume, of the thermocycling plate, relative to the initial fluidpassage. The distance between the second and third partitions can beless than two times greater, from about two times greater to about 200times greater, from about five times greater to about 150 times greater,from about 10 times greater to about 100 times greater, from about 25times greater to about 75 times, or more than 200 times greater than thedistance between the first and second partitions. The volume or lengthof the main fluid passage relative to the initial fluid passage can besimilarly varied.

When the initial fluid passage has a tortuous shape, it can comprise aplurality of cycles between the first partition and the secondpartition. A plurality of initial straight members, and a plurality ofinitial turn members joining the initial straight members at the firstand second partitions can be provided. The main fluid passage cancomprise a plurality of main straight members, and a plurality of mainturn members joining the main straight members at the second and thirdpartitions. Individual main straight members can have any lengthrelative to the individual initial straight members, but are generallylonger than the latter. Individual main straight members can have alength less than two times greater, from about two times greater toabout 200 times greater, from about five times greater to about 150times greater, from about 10 times greater to about 100 times greater,from about 25 times greater to about 75 times, or more than 200 timesgreater than the individual initial straight members. “Straight” membersare so named to distinguished them from turn members, however, thestraight members need not be completely straight nor do the turn membersneed to be curved. Straight members, turn members, or both can becurvilinear, rectilinear, or both. The back and forth path of the fluidpassageway can act as a counter-flow heat exchanger to improve thermalstability.

The main fluid passage, the initial fluid passage, or both can compriseany number of cycles between their respective partitions or otherwisesuitably orientated. For example, the main fluid passage can comprisefewer than 2 cycles, from about 2 cycles to about 2,000 cycles, fromabout 5 cycles to about 500 cycles, from about 10 cycles to about 100cycles, from about 15 cycles to about 75 cycles, from about 25 cycles toabout 50 cycles, about 88 cycles, or greater than 2,000 cycles betweenthe second partition and the third partition. The initial fluid passagecan comprise fewer than 2 cycles, from about 2 cycles to about 200cycles, from about 5 cycles to about 150 cycles, from about 10 cycles toabout 100 cycles, from about 15 cycles to about 75 cycles, from about 25cycles to about 50 cycles, about 40 cycles, or greater than 200 cyclesbetween the first partition and the second partition. The number ofcycles can correspond to the number of paths of the fluid passageway, inparticular, the main fluid passage, through the temperature zones.

In accordance with the present teachings, FIG. 8A shows a frontperspective view of a first thermocycling plate 304, and FIG. 8B is arear perspective view of the same plate. In FIG. 8C is shown a secondthermocycling plate 308, which is a mirror image of thermocycling plate304, with the rear perspective view shown in FIG. 8D. The firstthermocycling plate can have a plurality of corners, for example, fourcorners, 414, 418, 422, 426, bounding a plurality of edges, for example,four edges, 430, 434, 438, and 442. These corners and edges can define afirst face 502 and a second face 506. First, second, and thirdpartitions, 402, 406, and 410 are also provided.

In FIG. 8A, an inlet 388 and an outlet 392 are shown in fluid connectionwithin a first thermocycling plate 304. A fluid passageway 396 isconstructed through first thermocycling plate 304 in a generallyserpentine fashion in the form of a fluid passageway 396. Fluidpassageway 396 begins at inlet 388 and proceeds into an initial loopsector 400. The initial loop sector 400 falls within a hot start region404 that corresponds to the upper portion of the first static heatingzone 324 of first heating block 312. Fluid passageway 396 then entersinto a main cycling loop area 408, which corresponds to most of thefluid passageway 396 in first thermocycling plate 304. The upper part ofthe main cycling loop region 408 falls within a denaturing region 412.Denaturing region 412, along with hot start region 404, both line up andcorrespond to first static heating zone 324 of first heating block 312.The lower part of main cycling loops 408 corresponds to and is alignablewith second static heating zone 328 of first heating block 312. Maincycling loop region 408 can be segmented into a plurality of mainsectors. For example, in FIG. 8A, main cycling loop region 408 isdivided into a first main sector 420, a second main sector 424, a thirdmain sector 428, and a fourth main sector 432. An examination of themain cycling loop region 408 shows that the fluid passageway 396comprises both straight members 436 and turn members 440. An analogousconfiguration also can apply to initial loop sector 400.

Second (complementary) thermocycling plate 308, shown in FIG. 8C, hasanalogous components to first thermocycling plate 304. The secondthermocycling plate can have a plurality of corners, for example, fourcorners, 470, 474, 478, 482, bounding a plurality of edges, for example,four edges, 486, 490, 494, and 498. These corners and edges can define afirst face 530 and a second face 534. First, second, and thirdpartitions, 458, 462, and 466 are also provided. There are inlet 444,outlet 448, and fluid passageway 452. An initial loop sector 456 isprovided that is situated in a hot start region 460. Fluid passageway452 is next situated in the form of main cycling loop region 464 whichcorresponds to both a denaturing region 468 and its upper part and aannealing/extension region 472 in its lower part. As an example, maincycling loop region 464 is shown divided into first, second, third, andfourth main sectors 476, 480, 484, and 488. As was the case with fluidpassageway 396, fluid passage 452 comprises both straight members 492and turn members, or curved members, 496. FIGS. 8B and 8D show reverseperspective views of the thermocycling plates 304 and 308 shown in FIGS.8A and 8C, respectively. When two or more thermocycling plates areemployed, their respective fluid pathways can be joined to increase thetotal length of fluid pathway as well the number of cycles achievablewhen performing a thermocycling method such as PCR.

Thermocycling plates having different number and configuration ofsectors can be selected. For example, a thermocycling plate having adifferent number of main sectors can be selected. In another example,the number or length of straight members within sectors can be varied toachieve desired enhancement or sequencing performance. As illustrated inFIGS. 24A-24F, the length of straight members can be varied withinsectors or between sectors. For example, some straight sections within asector can be shorter, while other straight members can extend to thefull extent of the sector. As illustrated in FIG. 24D, some sectors canhave long straight members, while other sectors within the thermocyclingplate can have short straight members. As illustrated in FIG. 24B, thelong straight members within a sector can be adjacent one another, whilethe short straight members within the sector can be adjacent oneanother. Alternatively, the longer straight members can be separated byshort straight members, as illustrated in FIG. 24A or FIG. 24C.

Each of the illustrated plates can be used as either a first or secondthermocycling plate. In a particular example, the thermocycling plateillustrated in FIG. 24E can be used as a first thermocycling plate,while the plate illustrated in FIG. 24F can be used as a secondthermocycling plate.

In an alternative example, a single thermocycling plate can includefluid passageways molded into both sides of the plate. Flat faceplatescan be applied on either side of the double-sided thermocycling plate todefine the fluid passageways on both sides of the thermocycling plate.Alternatively, films can be applied to both sides of the thermocyclingplate to complete the formation of the fluid passageways. For example,as illustrated in FIG. 34 and FIG. 35, a single thermocycling plate1800, illustrated in cross-section, includes molded channels 1802, 1804,1806, and 1808 disposed on either side of the thermocycling plate 1800.When films 1814 and 1816 are attached to opposite sides of thethermocycling plate 1800, fluid passageways are defined.

In a particular example, and input port 1810 receives fluid, and directsthe fluid on one side of the thermocycling plate 1800. After traversingboth sides of the thermocycling plate 1800, the emulsion can exits port1812. In a particular example, the design the fluid passageways moldedwithin each side of the thermocycling plate 1800 can comply with one ormore features of the above-described thermocycling fluid passageways.

The thin films 1814 or 1816 disposed on opposite sides of thethermocycling plate 1800 can be formed of thin films having one or morelayers. In an example, the thin-film can include polymeric materials,such as polyolefins, polyesters, fluoropolymers, silicone, polyimide,polyamide, polycarbonate, or any combination thereof. In anotherexample, the thin-film can be formed of metallic material such asaluminum. In a further embodiment, the film can include more than onelayer such as layers of polyolefin, polyester, and aluminum. The films1814 and 1816 can be flexible. Further, such films 1814 and 1816 canhave a thickness in a range of 5 μm to 1000 μm, such as a range of 5 μmto 100 μm, a range of 5 μm to 50 μm, or even a range of 10 μm to 25 μm.Alternatively, the films 1814 or 1816 can be replaced with metallic orplastic sheets having a thickness greater than 1000 μm. The films 1814or 1816 can be secured to the thermocycling plate 1800 by a heat bond.Alternatively, an adhesive can be utilized to secure the films 1814 and1816 to the thermocycling plate 1800.

In an example, a thermocycling plate, when inserted into the systemconnects to existing tubing connecting an inlet to an outlet of theemulsifier and an outlet to a centrifuge. In a particular example, theamplified emulsion is transferred to the centrifuge in a fluid line alsoused to transfer a surfactant solution to the centrifuge.

In an alternative example, the thermocycling plate is a disposable platethat can incorporate a disposable flow path extending to a centrifuge.The thermocycling plate includes an inlet to receive an emulsion and anoutlet coupled to a disposable flow line to interface with a centrifuge.For example, as illustrated in FIG. 39, a thermocycling plate 3902 canbe coupled to an emulsifier 3904. In addition, the thermocycling plate3902 is coupled to a tube 3906. In a particular example, the tube 3906is integrated with the thermocycling plate 3902 prior to inserting thethermocycling plate 3906 into the system. The tube 3906 is to transferthe amplified emulsion to the centrifuge. In a particular example, thetube 3906 can extend through a pinch valve (not illustrated). Forexample, when installing the thermocycling plate 3902, the tube 3906 canbe threaded through a pinch valve. While the tube 3906 is illustrated asextending from an outlet at the lower end of the thermocycling plate3902, the tube 3906 can extend from an outlet near the top of thethermocycling plate 3902.

In one exemplary method of operation, emulsion from the emulsiongenerator 3904 is provided to the plate 3902. In a continuous manner,the emulsion flows through different temperature zones of the plate 3902based on the fluid path defined by the plate 3902. The emulsion flowsfrom the plate 3902 through the tube 3906 to an emulsion breakingcentrifuge.

In another exemplary method of operation, the emulsion from the emulsiongenerator 3904 is provided to the plate 3902. When the emulsion iswithin the plate 3902, flow can be halted and an optional pinch valve ontube 3906 can be closed. The plate 3902 can be thermocycled, causing atleast portions of the plate 3902 to cycle through differenttemperatures. Once the thermocycling protocol is complete, the optionalvalve can be opened and flow can be continued. The emulsion exits theplate 3902 through the tube 3906.

In a further exemplary method of operation, the emulsion from theemulsion generator 3904 is provided to the plate 3902. When the emulsionis within the plate 3902, flow can be halted and an optional pinch valveon the tube 3906 can be closed. The plate 3902 can be held at a constanttemperature for a period of time. Once the thermal protocol is complete,the optional valve can be opened and flow can be continued. The emulsionexits the plate 3902 through the tube 3906.

The thermocycling plate 3902 can have a path design as described above.Alternatively, the thermocycling plate 3902 can have a pathway asillustrated in FIG. 40. While the path of FIG. 40 includes verticalstraight sections, the path for the emulsion in the thermocycling plate3902 can have a horizontal path, including horizontal straight sections.In a further alternative, the thermocycling plate 3902 can have acircular or spiral path or can be replaced with a tube having a windingspiral or coiled configuration.

In an example, the tube 3906 can terminate in a needle or cannula. Theneedle or cannula can interface with an adapter connected to acentrifuge lid. Amplified emulsion can flow through the tube 3906,through the adapter and into a centrifuge, such as a slinger of thecentrifuge. For example, FIG. 38 includes an illustration of an adapter3800 to receive a needle or cannula 3808 attached to a tube 3806 with afastener 3810, such as a clip, a clamp, or a fastening material. Theadapter 3800 is secured in a lid 3804 of a centrifuge. For example, theadaptor 3800 can screw into an opening in the lid. In particular, theadapter can be positioned within the lid 3804 so that a distal end 3812of the needle or cannula 3808 is disposed over a slinger of thecentrifuge to distribute an emulsion into tubes on the rotor of thecentrifuge.

The adaptor 3800 can include an outer casing 3802, which defines acavity 3830 and through port 3826. A carriage 3814 is disposed withinthe cavity 3830 and is secured in place by a retaining ring 3818. Thecarriage 3814 includes a recess 3816 into which the retaining ring 3818extends. The recess 3816 allows the carriage 3814 to travel within thecavity 3830 of the adaptor 3800 relative to the retaining ring 3818. Thecarriage 3814 can include another recess for receiving a motivator 3820,such as a spring. The motivator 3820 provides force to motivate thecarriage 3814 away from the centrifuge lid 3804, in an upward directionin the illustrated example of FIG. 38. The carriage 3814 further definesa bore 3828 through which the needle or cannula 3808 can traverse. Theadaptor 3800 further includes a packing 3822 disposed to engage theneedle or cannula 3808 as it travels through the carriage 3814.

When the needle or cannula 3808 is inserted toward the centrifuge lid3804, through the bore 3828 of the carriage 3814, through the packing3822, and through the through port 3826, the needle or cannula 3808 isengaged by the packing 3822 which also engages the carriage 3814, movingthe carriage 3814 toward the centrifuge lid 3804, for example, to theextent permitted by the retaining ring 3818. As the needle or cannula3808 moves through the adaptor 3800, the distal end 3812 of the needleor cannula 3808 can contact a slinger or other apparatus of thecentrifuge, defining a lower boundary of movement for the distal end3812 of the needle or cannula 3808.

When the pressure or force driving the needle or cannula 3808 into theadaptor 3800 is released, such as when the distal end 3812 of the needleor cannula 3808 reaches a lower boundary, the motivator 3820 moves thecarriage 3814 away from the centrifuge lid 3804 or upward as illustratedin FIG. 38. Since the packing 3822 engages the needle or cannula 3808and the carriage 3814, when the carriage 3814 moves away from thecentrifuge lid 3804, the distal end 3812 of the needle or cannula 3808moves a short distance away from its lower boundary. Such movement canprevent blockages caused by the distal end 3812 of the needle or cannula3808 if it were to be in contact with a lower boundary.

The adaptor 3800 can further include a port 3824 for receiving a washsolution or surfactant solution supply. The port 3824 is in fluidcommunication with the through port 3826. When the needle or cannula3808 is engaged with the adaptor 3800, the through port 3826 can definean annulus extending through the through port 3826 for passing fluid tothe centrifuge.

The cross-sectional area of the fluid passageway of the thermocyclingplate can be constant, variable, or both, throughout its length or inwithin particular segments or regions. Cross-sectional area can bevaried for any suitable objective such as heat transfer or fluidvelocity. The present teachings have found that decreasing thecross-sectional area in turn members relative to adjacent straightmembers can increase the local velocity of the fluid passing through thefluid passageway and prevent or alleviate accumulation or queuing ofparticles or other bodies, for example, aqueous reactor droplets in an awater-in-oil emulsion. The turn members so configured can be thosemembers located along that partition that lies closer to the bottom ofthe thermocycling plate, depending how the plate is orientated, forexample, if the third partition is at the bottom (generally the case) orif the first partition is at the bottom. If the second edge or fourthedge of the thermocycling plate is at the bottom, the straight memberscan be configured to have a cross-sectional area less than that of theturn members.

At least one initial turn member or main turn member can have an averagecross-section area less than an average cross-section area of anadjacent initial straight member or adjacent main straight member. Aplurality of the initial turn members along the second partition caneach have an average cross-sectional area less than the averagecross-sectional area of an adjacent initial straight member. A pluralityof the main turn members along the third partition can each have anaverage cross-sectional area less than the average cross-sectional areaof an adjacent initial straight member. A plurality of the initial turnmembers along the first partition can each have an averagecross-sectional area less than the average cross-sectional area of anadjacent initial straight member. A plurality of the main turn membersalong the second partition each have an average cross-sectional arealess than the average cross-sectional area of an adjacent initialstraight member. Changes in cross-sectional area can be achievedabruptly or gradually, for example, by way of tapering the fluidpassageway. The cross-section can comprise one or more shapes. Examplesof shapes include circles, ellipses, squares, rectangles, and the like.The number and length of straight elements in the fluid passageway canbe determined by a person skilled in the art based on parameters such asthe desired number of reaction cycles and the fluid velocities.

FIGS. 9A and 9B show two embodiments focusing on the straight members436 and 492 as well as turn members 440 and 496. In FIG. 9A, a bottomregion 500 is shown that could correspond to a bottom region found ineither the main cycling loop region 408 or the initial loop sector 400.Bottom region 500 has a midpoint 504. Adjacent first straight member 508and second straight member 512 define in part the main passage 516 ofthe fluid passageway 396. Midpoint cross-section 520 at midpoint 504 ofbottom region 500 can be figured to have a cross-sectional area which isless than that of main passage cross-sectional area 524. By having themidpoint cross-sectional area less than the main passage cross-sectionalarea, the flow at midpoint 504 and adjacent regions is increasedrelative to the flow in main passage 516. An increase in flow at bottomregion 500 is desirable as a counter to gravity and the tendency ofmicroreactor liquid aqueous droplets in the water-in-oil emulsion thatpasses through fluid passageway 396 to accumulate at bottom regions.FIG. 9B shows an alternative embodiment bottom region 528 that employs arectilinear cross-sectional design as opposed to the curvilinearcross-sectional design of bottom region 500. Again, the cross-sectionalarea of the main passage, here 552, is greater than the midpointcross-sectional area, here 548, at midpoint 532 in order to achieve adesirable increase in flow. First straight member 536 and secondstraight member 540 correspond to the analogous members 508 and 512shown in FIG. 9A, as does main passage 544 to main passage 516.

The thermocycling plates of the present teachings can be constructedfrom any material or combination of materials. Examples of suitablematerials include plastics, glass, ceramics, metals, and the like.Examples of suitable plastics can include one or more of polypropylene,polycarbonate, polyimide, silicone, fluoropolymer, polyamide,polyvinylchloride, or any combination thereof. The thermocycling platecan be of any shape. The plate can be transparent, translucent, oropaque, or any combination thereof. The thermocycling plates can bemanufactured using any method. For example, the slab housing can be madeus of two complementary panels, each supplying one of the two respectivefaces of the slab housing, which are joined together with the facesfacing outwards. The thermocycling plates can also be constructed asdescribed in U.S. Patent Application Publication No. US 2008/0280331 A1.The present teachings also include pairs or sets of the thermocyclingplates. In a given pair or set, the individual thermocycling plates caninclude identical plates, non-identical plates, or both. For example, apair or set of plates can include thermocycling plates of the inventionthat are mirror images of one another. Plates can be configured forfluid connection with one another.

The thermocycling plates can be provided with any suitable dimensions.For example, the thermocycling plate can be slightly larger than astandard 96-well plate. The average diameter of the fluid passageway canbe less than about two times, from about two times to about 2,000 times,from about 5 times to about 1,500 times, from about 10 times to about1,000 times, from about 50 times to about 500 times, from about 100times to about 250 times, or greater than 2,000 times the diameter ofaqueous reactor droplets in a water-in-oil emulsion.

The thermocycling subsystem can be insulated using any means ormechanism. For example, a gasket can be used for insulating purposes.The gasket can be adjacent to at least three sides of each of the firstthermocycling plate and the first heating block. The gasket can beadjacent to at least three sides of each of the first thermocyclingplate, the second thermocycling plate, the first heating block, and thecomplementary heating block. The gasket can be constructed using anysuitable material or materials, for example, a rubber. The gasket cancomprise a compliant membrane.

FIG. 10A shows a gasket in accordance with the present teachings. Gasket556 can comprise a top section 560, a first lateral section 564, and asecond lateral section 568. First and second lateral sections 564 and568 can comprise, respectively, first lateral base 572 and secondlateral base 576. In a variation on the gasket 556 shown in FIG. 10A,first lateral base 572 and second lateral base 576 can be extended intoan optional bottom section 580 analogous to top section 560. Top section560, lateral first and second lateral sections 564 and 568, andoptionally bottom section 580 surround and define an aperture 584. Theaperture may be configured to allow insertion of thermocycling subsystem300 or 320. Depending on whether a first thermocycling subsystem 300 orsecond subsystem 320 is employed, lateral width 588 of gasket 556 can bevaried. Lateral width can also be varied independent of the particularthermocycling subsystem employed.

FIG. 10B shows gasket 556 with thermocycling subsystem 300 (or 320)inserted into aperture 584 of gasket 556. Resulting assembly 592includes both gasket 556 and thermocycling subsystem 300 (or 320).Visible are both the first and second heating blocks 312 and 316, alongwith tubing 596 extending from inlet 444 and outlet 448 through accessrecess 384 of heating block 316.

While the above described thermocycling system can operate in acontinuous manner with an emulsion flowing continuously from theemulsifier and into the thermocycling plate and through thethermocycling plate to a centrifuge, as the emulsion flows through thethermocycling plate, the emulsion flows between sections of differenttemperature, facilitating PCR.

Alternatively, the thermocycling plate can be used in a batch orsemi-batch mode. The emulsion can flow into the thermocycling plateuntil the emulsion is within the tortuous path of the thermocyclingplate. While the emulsion flows to the thermocycling plate, the plate isheld at a constant temperature. The heat plates can be replaced with athermocycler. Once the emulsion is within the plate, flow can be stoppedand the plate temperature cycled to facilitate PCR. When PCR iscomplete, the emulsion can be pumped from the plate to the centrifuge.

FIGS. 11A and 11B show an emulsion thermocycling system 600, includingthermocycling subsystem 300 in an open and closed configuration,respectively. Such a system can be used to generate and recover a PCRproduct. FIG. 11A shows thermocycling subsystem 300 in an openconfiguration in the right perspective view of emulsion thermocyclingsystem 600. A thermocycling system 600 has a top surface 604 residing ona housing. The opening of thermocycling subsystem 300 can be achieved byusing a lever 608. Lever 608 includes a lateral handle portion 612mounted between a first arm 616 and a second arm 620. Lever 608 isconfigured so that first arm 616 and second arm 620 can engage inrespective first track 624 and second track 628. When lever 608 ispulled back relative to a front of the emulsion thermocycling subsystem,an opening 632 is provided between first and second heating blocks 312and 316. FIG. 11B, showing a left perspective view of the emulsionthermocycling subsystem shown in FIG. 11A, depicts thermocyclingsubsystem 300 in a closed configuration. The closed configuration isachievable by pulling or pushing forward lever 608 so that first heatingblock 312 and second heating block 316 are brought together andimmediately adjacent to each other so as to close the opening 632 thathas been shown in FIG. 11A. Lever 608 can be configured and arranged inany appropriate configuration. For example, a lever mechanism asdescribed in U.S. Pat. No. 6,896,849 B2. A latch or locking mechanismfor the lever can also be incorporated into thermocycling subsystem 300and emulsion thermocycling system 600.

In accordance with the present teachings, a method of thermocycling isprovided. The method can comprise one or more of the following steps,the order of which can be varied, or one or more steps can be repeated.A source solution can be passed through a thermocycling plate comprisinga plurality of regions. A hot start region corresponding to an initialfluid passage of the thermocycling plate can be heated and adenaturation region corresponding to a portion of a main fluid passageproximal a second partition of the thermocycling plate can be heated toa first temperature or temperature range. An annealing/extension regioncorresponding to a portion of the main fluid passage proximal a thirdpartition of the thermocycling plate can be heated to a secondtemperature or temperature range. The first temperature can be higherthan the second temperature, and the first temperature range can behigher than second temperature range.

The sample fluid passed through the fluid passageway can comprise awater-in-oil emulsion comprising a plurality of aqueous polymerase chainreaction (PCR) reaction droplets. Such droplets and water-in-oilemulsion can be formed using the emulsion generation techniquesdescribed herein. When using the thermocycling plate for PCRamplification, the method can comprise annealing a sample nucleic acidin the reactor droplet to a template in the reactor droplet, extendingthe sample nucleic acid to form a double-stranded nucleic acid,denaturing the double-stranded nucleic acid, and repeating such stepsuntil a desired amplification can be achieved. Sample (source) fluid canbe sent from the thermocycling subsystem to a centrifuge subsystem. Forexample, PCR product in the sample solution can be sent from thethermocycling plate outlet to a centrifuge for recovery of the PCRproduct. The method can also include stabilizing the thermocycling platewith an oil phase prior to passing an emulsion through the fluidpassageway. The fluid can be passed through the fluid passageway at anydesirable rate or acceleration. For example, the fluid can be passedthrough the fluid passageway at a rate of less than 0.001 mL/min, fromabout 0.001 mL/min to about 1 L/min, from about 0.01 mL/min to about 500mL/min, from about 0.1 mL/min to about 250 mL/min, from about 0.25mL/min to about 100 mL/min, from about 0.5 mL/min to about 50 mL/min,from about 1.0 mL/min to about 10 mL/min, or greater than 1 L/min. Fluidcan be passed through continuously or bath-wise. Aqueous reactordroplets in the oil-phase can pass through the fluid passageway in anymanner. While, such passage can comprise a single file passage, singlefile passage is not necessary, and simultaneous passage of more than onedroplet through a given cross-section is permitted.

Centrifugation Subsystem and Emulsion Breaking

In accordance with the present teachings, a centrifuge subsystem isprovided that is suitable for integration into an emulsion PCR system orequivalent device. The centrifuge can comprise a centrifuge housing, amotor comprising a rotor axle mounted in the housing motor aperture; anda rotor mounted on the rotor axle or otherwise operably connected to aspinning means. The centrifuge housing can comprise sidewalls extendingfrom a base portion to a top portion comprising a top housing rimsurrounding a top housing aperture. A lid operably associable with thetop housing rim can be provided. The housing can further include ahousing bottom recess extending from the base to an interior of thecentrifuge housing and comprising bottom recess sidewalls and a bottomrecess ceiling. A housing basin can be defined by the top housing rim,top housing aperture, and housing basin sidewalls surrounding areceiving platform. The housing can also provide a housing motoraperture configured to accept a motor through the bottom recess ceilingand receiving platform. The housing can be constructed of any suitablematerial or combination of materials, for example, those materialsdescribed herein for the collection tubes, slinger, and rotor. Any orall components of the centrifuge subsystem can be transparent,translucent, or opaque, or any combination thereof.

The centrifuge can be used with or without a lid. The lid can beconnected to the centrifuge housing using any suitable manner. Thehousing or lid can be secured with threading to allow the lid to bescrewed on and off the centrifuge housing. A clamp can be provided onthe lid housing or centrifuge housing to allow the lid to be attachedand detached from the centrifuge housing. The lid housing or centrifugehousing can be provided with a gasket or other sealing mechanism toallow for a tight fit between the lid and centrifuge sidewalls. The lidcan be attached to the centrifuge housing using one or more hinges. Forexample, a housing hinge assembly can be employed that comprises ahousing hinge plate operatively connectable to the centrifuge housing.The housing hinge plate can include a top plate portion, first andsecond hinge receiving arms extending from the top plate portion andcomprising respective first and second hinge axle apertures. A hingeaxle can extend from the first and second hinge axle apertures andpassing through a lid hinge portion extending from the lid.

Optionally, the lid can be a locking lid. A locking mechanism can beassociated with the centrifuge to engage the lid, particularly duringoperation of the centrifuge. In an example, the locking mechanism canautomatically engage and lock the lid when the centrifuge is inoperation. In another example, the lid can be manually locked. In afurther example, the locking mechanism can provide a sensor to determinewhether the lid is secure in a closed position before operation of thecentrifuge is permitted.

FIG. 12A is a front perspective view of an embodiment in accordance withthe present teachings. A centrifuge subsystem 700 comprises a centrifugehousing 704. The centrifuge housing 704 includes centrifuge housingsidewalls 708 that extend to a base portion 712. Base portion 712 cancontain a flange 716 that can provide mounting apertures 720 along aflange perimeter 724. Mounting apertures 720 allow the centrifugesubsystem 700 to be mounted in an emulsion thermocycling system or thelike. Top portion 728 of centrifuge housing 704 provides a foundationfor a lid 732 when in a closed configuration as shown in FIG. 12A. Lid732 is constructed with a lid housing 736 that provides a top lidsurface 740 and a bottom lid surface 742 through which a central lidaperture 744 is located. Lid 732 is attached to centrifuge housing 704through a hinge 748.

A side perspective view of centrifuge subsystem of 700 is shown in FIG.12B. This view of centrifuge subsystem 700 affords a more detailedperspective of hinge 748. Hinge 748 comprises a lid hinge portion 752that is received by a first hinge receiving arm 756 and second hingereceiving arm 760, which are a part of centrifuge housing 704.

First and second hinge receiving arms 756 and 760 comprise respectivelyfirst axle receiving aperture 764 and second receiving aperture 768. Ahinge axle 772 can be placed through the first and second receivingapertures to secure lid hinge portion 752. First and second hingereceiving arms 756, 760 are part of a housing hinge assembly 776, whichis described in greater detail in respect to FIG. 12C. In FIG. 12B,visible in centrifuge housing sidewall 708 is a housing drain aperture780. Housing drain aperture 780 allows passage of a tubing for drainageof fluids from within centrifuge subsystem 700.

FIG. 12C displays a rear perspective view of centrifuge subsystem 700.The rear view of centrifuge subsystem 700 affords visibility of housinghinge assembly 776. Housing hinge assembly 776 comprises a housing hingeplate 784. Housing hinge plate 784 comprises hinge plate mountingapertures 788. Hinge plate mounting apertures 788 allow the housinghinge plate to be connected to the centrifuge housing 704. While FIG.12C shows the housing hinge plate as discrete element and connected tothe centrifuge housing, other embodiments allow the lid to be directlyformed with the centrifuge housing 704. A power supply line aperture 792allows entry of a power supply line 796 through housing sidewall 708 tosupply power to a motor configured to drive a rotor of the centrifugesubsystem 700. Use of a power supply line aperture is optional as powercan be supplied to the motor by other means. For example power could besupplied from below the motor through a power supply line emanating froman emulsion thermocycling system or apparatus.

A bottom perspective view of centrifuge subsystem 700 is shown in FIG.12D. Centrifuge housing 704 is inset to provide a housing bottom recess800. Housing bottom recess 800 comprises bottom recess sidewalls 804 anda bottom recess ceiling 808. The bottom recess ceiling provides ahousing motor aperture 812 through which a motor 816 can be inserted.Motor 816 comprises a motor housing 820 as well a motor rotor axle 824.

Any suitable motor can be utilized as part of the centrifuge such thatit can provide the desired angular velocity and acceleration to thecentrifuge rotor. Motor control can be one directional or can involve afeedback mechanism. Means and mechanisms can be provided to measure orcontrol at least one of the number of revolutions, velocity,acceleration, and braking Control can be voltage, current, or resistancebased. Any suitable centrifuge construction, power, and control can beemployed, for example, as described in U.S. Pat. Nos. 3,990,633,4,070,290, 4,822,331, 5,342,280, and U.S. Pat. No. 6,879,262 B1, whichare incorporated herein in their entireties. A pattern of contrastalternation, for example, black and white, can be provided on theunderside of the rotor in operable combination with an optical detector.The motor can be provided with a housing that contains the components ofthe motor. The motor housing can include any device or mechanism toallow for mounting or other connection to the centrifuge housing. Themotor can comprise a rotor axle on which the rotor can be mounted orotherwise connected. Multiple motor can be employed. The motor can beconfigured for use with direct current (DC), alternating current (AC),or both. The angular direction of the motor can be adjust to move eitherclockwise or counter-clockwise, or both. Any suitable rotor can beemployed such as the rotor described herein in accordance with thepresent teachings.

FIG. 13A shows a side perspective view of centrifuge subsystem 700 in anopen configuration. Lid 732 is in the open configuration such that theinterior of the centrifuge 700 can be seen as well as the top housingrim 828 on which lid 732 rests while in a closed configuration. A rotor832 having a rotor housing 836 is visible through top housing aperture840 defined by top housing room 828. Centrifuge 700 provides a housingbasin 844 as visible and defined by the top housing room 828. Housingbasin 844 provides basin sidewalls 848 as well as a receiving platform852. Receiving platform 852 comprises motor mounting apertures 856 aswell as a central rotor axle aperture 860.

FIG. 13B shows the housing basin 844 and rotor 832 in greater detail.Rotor 832 and its rotor basin 864 comprise rotor basin sidewalls 868 androtor basin floor 870. And a rotor top rim around the periphery andupper end of rotor basin sidewalls 868. Rotor basin sidewalls 866provide at least one collection tube receptacle. As shown in FIG. 13B,rotor basin sidewalls 868 provide first tube receptacle 876 and secondtube receptacle 880. Any number of tube receptacles can be provided inrotor basin sidewall depending in part on the size of the tubes to beinserted and the overall size and surface area of the rotor basinsidewall 868. Rotor top rim 872 has an inner perimeter 873 and outerperimeter 874. Alongside the respective tube receptacles and provided inrotor top rim 872 are collection tube exit channels receptacles. As seenin FIG. 13B, first tube exit channel receptacle 884 and second tube exitchannel receptacle 888 are provided respectively with first and secondreceptacle grooves 886, 890. At the center of the rotor basin 864 is afluid distribution device or slinger receptacle 892 comprising slingerreceptacle sidewalls 894.

A fluid collection tube is provided comprising a main tube body and atube extension. The main tube body can comprise a main body sidewallsurrounding a tube interior comprising a tube opening at a first end,and a second end distal to the first end providing a sealed base. Thetube extension can comprise a tube extension sidewall defining a fluidexit channel in fluid communication with the tube interior through atube channel inlet proximal to the tube opening and extending to a tubechannel outlet distal to the tube opening. The fluid collection tube canfurther comprise a tube lip disposed about the perimeter of the tubeopening and allowing fluid communication of the tube exit channel withthe tube interior. An optional tube buttress disposed between the tubeextension sidewall and the main tube sidewall provides further supportand rigidity. The tube extension sidewall and the main tube sidewall canbe positioned at any angle relative to one another, from about 15° toabout 90°, from about 1.0° to about 80°, from about 5.0° to about 75°,from about 10° to about 65°, from about 20° to about 60°, from about 25°to about 50°, from about 30° to about 45°, from about 30° to about 60°,from about 40° to about 50°, or greater than about 90° relative to eachother.

The fluid collection tube can be provided in any suitable shapeincluding the main tube body and the tube extension. For example, themain tube body can comprise a generally cylindrical portion proximal thefirst end, a conically tapered portion proximal the second end, and arounded second end. For example, the tube exit channel and tubeextension sidewall can have a U-shaped cross-section along alongitudinal axis. The average cross-sectional area of the exit tubechannel can be less than, equal to, or greater than the averagecross-sectional area of the tube interior. The average cross-sectionalarea of the exit tube channel can be less than about 95%, less thanabout 90%, less than about 75%, less than about 60%, less than about50%, less than about 40%, less than about 25%, less than about 20%, lessthan about 15%, less than about 10%, less than about 5%, or less thanabout 1% of the average cross-sectional area of the tube interior.

The fluid collection tube of the present teachings can optionallycomprise a cap or lid. The lid can be completely separable from the tubehousing or can be permanently joined to the housing, for example,through a flexible hinge. The cap can be configured to close off themain tube interior, the fluid exit channel, or both.

The fluid collection tube can be constructed from any suitable material.Suitable materials include metals, plastics, glass, ceramics, or anycombination thereof. Examples of suitable plastics includepolypropylene, polycarbonate, and polyvinyl chloride. The fluidcollection tube can be constructed to contain any desired volume. Thefluid collection tube can have a volume of less than about 1 μL, fromabout 1 μL to about 1 L, from about 10 μL to about 1 dL, from about 100μL to about 1 cL, from about 500 μL to about 50 mL, from about 1 mL toabout 25 mL, from about 2.5 mL to about 15 mL, from about 5 mL to about10 mL, or greater than about 1 L.

FIG. 14A shows a side perspective view of a collection tube 896 inaccordance with the present teachings. Collection tube 896 comprises atube housing 900 including a main tube body 904, comprising a tubeinterior 902, and a tube extension 906 comprising a fluid exit channel908. The length of the collection tube is defined by a first tube end912 and a second tube end 916. At first tube end 912, is a tube lip 920that can be engaged by rotor sidewall apertures tube receptacles 876,880. Fluid exit channel 908 has a length defined by a tube channel inlet924 and a tube channel outlet 928.

A top perspective view of collection tube 896 is shown in FIG. 14B. Inparticular an angle 932 is shown defined by the spacing of the sidewalls 910 of tube extension 906 from main tube body 904. An optionaltube buttress 930 is also shown between the main tube body and the tubeextension. Collection tube 896 is again visible in FIG. 14C. Tube inlet920 defines and surrounds a tube opening (aperture) 936 having a tubeopening center 938. At the middle of tube opening 936 is tube openingcenter 938.

A fluid distribution device, also referred to herein as a “slinger,” isprovided by the present teachings. The slinger can comprise sidewallsdefining a central channel comprising a first end, a central zone, and asecond end along a longitudinal axis. Sidewall lateral extensions, alsoreferred to as “wings” herein, can be provided, extending away from thesidewalls on either side of the central channel. The wings are useful inmating with and ensuring a secure connection with a fluid distributiondevice receptacle of a centrifuge rotor. The slinger can have any numberof spouts that can be correlated with the number of fluid collectiontubes to be used in conjunction with the slinger. For example, when twofluid collection tubes are employed, the slinger can include a firstspout extending from the central zone to the first end and terminatingat a first spout outlet, the sidewalls tapering along the central zonetoward the first spout; and a second spout extending from the centralzone to the second end and terminating at a second spout outlet, thesidewalls tapering along the central zone toward the second spout.

The slinger can be configured to mate with a fluid distribution devicereceptacle of a centrifuge rotor using any suitable configuration. Forexample, wings, as described herein, can be employed. Rather than use aninsertable/detachable slinger, a slinger can be employed that ispermanently or integrally associated with the rotor housing.

The slinger can be fabricated from any suitable material or materialssuch as those described herein for the collection tube. The slinger cancomprise one or more of the materials described herein for the fluidcollection tubes. The slinger can have any desired volume. The slingercan have a volume or less than about 1 μL, from about 1 μL to about 1 L,from about 10 μL to about 1 dL, from about 100 μL to about 1 cL, fromabout 500 μL to about 50 mL, from about 1 mL to about 25 mL, from about2.5 mL to about 15 mL, from about 5 mL to about 10 mL, or greater thanabout 1 L.

FIG. 15 is a plan view of a liquid distribution device or slinger 940.Slinger 940 comprises a slinger housing 944, which is shaped to provideslinger sidewalls 948, a slinger base 952, and slinger wings 956. Aslinger central channel 960 is defined by the slinger base 952 andslinger sidewalls 948 running the length of the slinger. At the centerof the slinger is a central zone 962. Well slinger 940 is shown having asingle central channel 960 additional central channels can be providedin other embodiments. First slinger spout 964 and second slinger spout968 comprise opposite sides and ends of the slinger central channel. Ateither end of slinger central channel 960 as well as the ends ofrespective first slinger spouts 964, 968 are first slinger spout outlet972 and second slinger spout outlet 976.

A centrifuge rotor is provided by the present teachings, which isparticularly suitable for separating water-in-oil emulsions and removingthe oil phase of such an emulsion. The centrifuge rotor can comprise arotor housing having a bisecting rotor axis perpendicular to a centralrotor axis, a rotor basin formed by the rotor housing, a rotor basinfloor, and a rotor basin sidewall lining the rotor housing basin andextending up toward a rotor top rim having an inner perimeter and anouter perimeter. The rotor can further comprise at least one collectiontube receptacle comprising an opening formed in the basin sidewall, andat least one tube extension receptacle having a grove formed in therotor top rim and extending from the inner perimeter to the outerperimeter. The sidewalls can comprise a substantially flat inset regionabout the collection tube receptacle and adjacent the tube receptacleopening.

The centrifuge rotor can be provided with any number of collection tubereceptacles and corresponding tube extension. Generally, an even numberof receptacles are provided on opposite sides of the rotor basin. Forexample, first and second tube receptacles positioned opposite eachother along the bisecting rotor axis; and first and second tubeextension grooves opposite each other along the bisecting rotor axis. Anodd number of receptacles can be utilized, and in such embodiments therotor can be balanced to account for the weight of any unpairedreceptacles. Balancing can also be provided for embodiments where aneven number of receptacles are provided but when not all receptacles arefitted with collection tubes or collection tubes of unequal volume orweight. The rotor can also comprise at least one liquid distributiondevice (slinger) receptacle extending from the rotor basin floor andhaving a distribution device receptacle longitudinal axis.

The collection tube receptacle opening and the tube extension groove aregenerally positioned at an angle relative to each other corresponding tothe angle of the tube extension sidewall relative to the main tube body.For example, the collect tube receptacle and the tube extension groovecan be from about 15° to about 90°, from about 1.0° to about 80°, fromabout 5.0° to about 75°, from about 10° to about 65°, from about 20° toabout 60°, from about 25° to about 50°, from about 30° to about 45°,from about 30° to about 60°, from about 40° to about 50°, or greaterthan about 90° relative to each other. The shape of the rotor sidewallcan be configured to accommodate a collection tube buttress. The fluiddistribution device receptacle can include a means for receiving orreversibly locking in place the slinger. For example, the slingerreceptacle can contain opposing sidewalls on either side of thedistribution device receptacle longitudinal axis that can cooperate withwings on the slinger.

The slinger receptacle can be provided in any suitable configurationwithin the centrifuge rotor. The bisecting rotor axis can be parallel ornon-parallel to the distribution device receptacle longitudinal axis.The axes can be positioned from about 0.01° to about 25°, from about0.05° to about 20°, from about 0.15° to about 15.0°, from about 0.25° toabout 10.0°, from about 0.5° to about 5.0°, from about 1.0° to about2.5°, less than about 0.01°, or greater than about 25° relative to eachother.

FIG. 16 is a top perspective view of rotor 832 as positioned in housingbasin 844. First collection tube 896 and second collection tube 898 aredisposed in respective first and second tube receptacles 876 and 880.Slinger 940 is positioned in slinger receptacle 892. Collection tubes896 and 898 each have respective tube openings 936 and 980, which inturn have respective tube opening centers 938 and 982. Cutting the rotorapproximately in half is a bisecting rotor axis 984, which can beperpendicular to central rotor axis 986, extending into and out of thepage. Along the slinger central channel 960, along the length of slinger940 and extending out either end is a slinger longitudinal axis 988.Slinger 940 is mounted in slinger receptacle 892 so that slinger 940 isoffset from the centers of the respective collection tubes 896, 898.Rather than the slinger spout outlet 972 being directly in line with thefirst tube opening center 938, there is an offset of and by an angle 992defined by the spacing of bisecting rotor axis 984 from slingerlongitudinal axis 988. Angle 992 can be varied as appropriate. Theoffset of slinger 940 from the respective tube opening centers accountsfor angular movement of the rotor such that when in motion any fluidexiting respective first slinger spout outlets 972, 976 will land at ornear respective tube opening centers 938, 932. The offset angle 992 canbe varied to account for changes in the angular speed of or accelerationfor the rotor 832.

When the centrifuge is run in accordance with the present teachings,fluid from the fluid collection tubes is expelled from the rotor throughthe tube exit channels. The expelled fluid can be accumulated,processed, or discarded using any suitable means or mechanism. Forexample a peripheral gutter can be employed. The peripheral gutter caninclude a gutter housing providing a top gutter surface, a bottom guttersurface, and gutter sidewalls extending between the top and bottomgutter surfaces along an outer gutter perimeter of the gutter housing. Agutter inlet can be located along an inner perimeter of the gutterhousing. The gutter can be connected to the centrifuge housing using anymeans or mechanism. For example, a gutter flange extending around thegutter outer perimeter and adapted for placement on the top housing rimcan be employed. The fluid collected in the peripheral gutter can bediscarded using any means or mechanism. For example, a gutter outlet canbe provided in the gutter housing along with a housing drainage aperturein the housing sidewall and a basin drainage aperture in the housingbasin sidewall. In such a configuration, a drainage tubing can beoperatively associated with the gutter outlet and pass through thedrainage apertures.

A peripheral gutter 996 is shown in perspective view in FIG. 17 inaccordance with various embodiments of the present teachings. Peripheralgutter 996 serves the function of collecting waste fluid from thecentrifuge when the rotor is in operation. Peripheral gutter 996comprises a gutter housing 1000 providing a top surface 1004, guttersidewalls 1008, and gutter base 1012. Gutter housing 1000 defines agutter interior 1016. Providing access to gutter interior 1016 is agutter inlet 1020. A gutter outlet 1024, in turn, is provided at one ormore points along the gutter sidewalls 1008 or gutter base 1012. Along atop outer gutter perimeter 1028 is provided a gutter flange 1032. Gutterflange 1032 is configured to allow placement and engagement on tophousing rim 828 of centrifuge housing 704.

FIG. 18A shows a top perspective view of centrifuge subsystem 700 asassembled with various components including peripheral gutter 996, firstand second collection tubes 896, 898, and slinger 940. A drainage tubing1036 is shown engaged with gutter outlet 1024 and can be passed throughbasin drainage aperture 782 and housing drain aperture 780. A sideperspective view of centrifuge subsystem 700 as assembled is also shownin FIG. 18B.

A cross-sectional view of the assembled centrifuge subsystem 700 isshown in FIG. 18C. Rotor 832 rests on and is connected to rotor axle 824of motor 816. First and second collection tubes 896, 898 are shownfilled with fluid comprising an oil/water interface 1040 separating anoil phase 1044 from an aqueous phase 1048. Collection tubes 896,898 cancomprise at least one mixing ball each in addition aqueous solution.Such bead or beads are located at the base or second tube end 916. Whenthe rotor is in angular momentum the oil phase can exit the collectiontubes through the respective exit tube channels and land in peripheralgutter 996.

The centrifuge of the present teachings is particular suitable for thebreaking of emulsions. The emulsion can be in form a sample, i.e.,source fluid. Any means or mechanism can be employed to deliver sourcefluid to the centrifuge. The sample fluid can be delivered through aninlet supply line (fluid supply line). When a lid is not employed, or isnot in a closed configuration, the fluid line can be delivered directlyinto the centrifuge through the top centrifuge opening afforded by thecentrifuge housing sidewalls. When a lid is employed and is in a closedconfiguration, the fluid supply line can pass through at least one of alid aperture and a housing aperture to gain access to the interior ofthe centrifuge. For example, the lid can comprise a housing with a toplid surface and a bottom lid surface, a lid aperture extending from thetop lid surface to the bottom lid surface. The lid aperture can belocated anywhere on the lid, for example, it can be centrally located.More than one lid aperture can be provided. The lid aperture can beconfigured to accept a fluid supply line and positioned above the fluiddistribution device when the lid is in a closed configuration. A fluidsupply source can be in fluid communication with the fluid supply line.A fluid supply pump can be configured to pump fluid from the fluidsupply source, through the fluid supply line and into the centrifuge.

Centrifuge subsystem 700 is shown in FIG. 19 in a closed configurationand operably connected to a fluid source. A lid aperture adapter 1052 isconnected to centrifuge subsystem 700 through central lid aperture 744.Inlet fluid line 1056 can pass either directly into centrifuge subsystem700 or through one or more of a lid aperture adapter and a fluid lineconnector 1060. Fluid line 1056 can originate from a fluid sample source1070. Fluid sample source 1070 can be supplied to centrifuge subsystem700 by means of a fluid sample source pump 1064. As shown in FIG. 19,pump 1064 is a syringe style pump. The present teachings allow for useof other types of pumps as well or in addition to a syringe style pump.For example, a peristaltic pump or diaphragm pump could be employed aspump 1064. Fluid line 1056 can originate from an emulsion subsystem 300.The sample fluid supplied by inlet fluid 1056 can comprise awater-in-oil emulsion. The water phase of the water and oil emulsion cancomprise microreactors that in turn can contain PCR product or products.

A method of recovering a polymerase chain reaction (PCR) product from awater-in-oil emulsion is provided in accordance with the presentteachings. The method can be performed using the centrifuge orcentrifuge components of the present teachings. The method can compriseone or more of the following steps. One or more step can be repeated oromitted. The order of the method steps can be varied. An example of sucha method is shown in FIG. 20 (1100). At least one collection tube isfilled with an aqueous solution (1110). At least one collection tube isinserted into a tube receptacle of a centrifuge rotor (1120). At leastone fluid distribution device (slinger) is inserted into a fluiddistribution device receptacle of the centrifuge rotor (1130). Samplefluid, which can comprise a PCR product in a water-in-oil emulsion, isfed into the fluid distribution device (1140). The centrifuge rotor isspun to deliver the sample fluid to the at least one collection tube(1150). The emulsion is partly or completely broken in the at least onecollection tube (1160). The centrifuge rotor motor can also be spun toremove an oil phase from the collection tube (1170). The method canfurther comprise collecting the oil phase in a peripheral gutter, thatis, the oil phase removed from the collection tube. The method canfurther comprise recovering PCR product from the at least one collectiontube (1180).

Any suitable aqueous solution can be employed in the collection tubesand generally comprises at least water. The aqueous solution can containa surfactant, a detergent, or both. A component that is both asurfactant and a detergent can be employed. Multiple surfactants ordetergents can be used. Any surfactant or detergent can be used, forexample, those described in U.S. Pat. No. 4,938,876, which is hereinincorporated in its entirety. For example, the aqueous solution cancomprise sodium dodecyl sulfate (SDS). The aqueous solution can comprisean alcohol, for example, ethanol. The surfactant, detergent, SDS,alcohol, or ethanol, alone, or in any combination can be present in theaqueous solution in an amount less than about 0.001 vol. %, from about0.001 vol. % to about 100 vol. %, from about 0.01 vol. % to about 95vol. %, from about 0.1 vol. % to about 90 vol. %, from about 0.5 vol. %to about 85 vol. %, from about 1.0 vol. % to about 80 vol. %, from about5.0 vol. % to about 75 vol. %, from about 10 vol. % to about 60 vol. %,from about 15 vol. % to about 50 vol. %, from about 20 vol. % to about40 vol. %, or from about 25 vol. % to about 35 vol. % of the totalvolume of the aqueous solution. An SDS solution can also be used as awash solution. The aqueous solution can comprise one or more salts toaid in the breaking of the emulsion. One or more acid or base can beadded to the aqueous solution to adjust pH to assist in breaking theemulsion.

Use of a mixing ball can aid in the recovery of PCR product laden beads.The at least one collection tube can contain at least one mixing ballcapable of capturing PCR product associated beads. The mixing ball canbe constructed from any suitable material, for example, one or morematerial described herein for constructing fluid collection tubes,slingers, and housings. Any number of beads can be recovered. Forexample, from about 1 bead to about 1 trillion beads can be recovered,from about 100 beads to about 100 million beads can be recovered, fromabout 1,000 beads to about 10 million beads, from about 10,000 beads toabout a million beads, from about 100,000 beads to about 500,000 beads,or more than 1 trillion beads can be recovered. Percent recovery ofbeads can be any desired percentage based on the number of beadssupplied to the centrifuge tube. Bead recovery can be quite high such as120 million beads recovered out of 140 million input. For example, beadrecovery can be less than 1.0%, from about 1.0% to about 100%, fromabout 5.0% to about 95%, from about 10% to about 90%, from about 15% toabout 85%, from about 20% to about 80%, from about 25% to about 75%,from about 30% to about 60%, or from about 40% to about 50% of inputbeads.

The fluid distribution device (slinger) can be inserted into acorresponding receptacle of the rotor so that at least one fluiddistribution outlet is displaced off center from a center of the atleast one collection tube, and the spinning occurs in the direction ofthe displacement. The degree of slinger displacement can be relative toat least one of an angular velocity and an angular acceleration of thespinning rotor. The degree of the displacement can result in the samplefluid entering the at least one collection tube at a center of the tube.The rotor can be spun at any desired speed. For example, the rotor canbe spun less than 1 rpm, from about 1 rpm to about 100,000 rpm, fromabout 10 rpm to about 60,000 rpm, from about 50 rpm to about 30,000 rpm,from about 100 rpm to about 10,000 rpm, from about 500 rpm to about5,000 rpm, or from about 1,000 rpm to about 2,500 rpm or more. Theacceleration of the rotor can be held constant, varied, or both. Forexample, the acceleration can be less than about 1 G, from about 1 G toabout 1,000 G, from about 3 G to about 300 G, from about 5 G to about100 G, from about 10 G to about 50 G, from about 15 G to about 30 G, orgreater than 1,000 G.

The contents of the at least one collection tube can be mixed oroscillated by alternating the direction of the spinning rotor.Oscillation can be employed to resuspend beads or particles. Thedirection of the rotor can be changed at any desired frequency. The rateof frequency can also be varied over time. For example, the direction ofthe rotor can be changed about every 1 millisecond, about every 10milliseconds, about every 100 milliseconds, about every 1 second, aboutevery 10 seconds, about every 30 seconds, about every minute, aboutevery 5 minutes, about 10 minutes, about every 15 minutes, about every20 minutes, about every 30 minutes, or about every hour.

According to various embodiments of the present teachings, amplified DNAfragments tethered to a particle or bead can be prepared. Device,systems, apparatuses, and methods are described herein relating to theamplified polynucleic acid tethered particles or beads. The method canbegin by forming an inverse emulsion comprising a plurality of aqueousdroplet microreactors encapsulated and separated from one another by acarrier fluid, for example, an immiscible oil or a fluorinated liquid.Each microreactor, or at least one of them, can contain a template bead,also referred to as a P1 bead or a primer 1 bead, and PCR ingredients.The amplification process may be referred to as a bead-based emulsionamplification. Beads along with DNA templates can be suspended in anaqueous reaction mixture (a microreactor mixture) and then droplets ofthe mixture can be encapsulated by the immiscible liquid in an inverse(water-in-oil) emulsion. The template DNA may be either bound to thebead prior to emulsification or may be included in solution in theamplification reaction mixture.

According to various embodiments, a method and system are provided forautomated sample preparation for sequencing applications. In someembodiments, bead-based emulsion amplification is performed uponformation of an emulsion which encapsulates aqueous droplets. Eachdroplet can contain a template DNA strand and a bead upon whichamplicons to be formed from the template DNA. The droplet can alsocontain a reagent mixture for enabling the amplification reaction. Theemulsion can comprise an inverse (water-in-oil) emulsion with theaqueous phase (e.g., the microreactor mixture) including the reagentmixture and the bead, and the carrier fluid including oil or othernon-aqueous liquid that is partially or completely immiscible with theaqueous phase.

According to various embodiments, an emulsion thermocycling subsystem isprovided. The thermocycling subsystem can comprise a first thermocyclingplate, as described herein, and a heating subassembly, as describedherein. The heating subassembly of the thermocycling system can alsocomprise a complementary heating block as described herein. The heatingsubassembly of the thermocycling system can also comprise a secondthermocycling plate. The thermocycling subsystem can be insulated usingany means or mechanism. For example, a gasket can be used for insulatingpurposes.

In some embodiments, a thermocycling plate is provided in accordancewith the present teachings. The thermocycling plate generally comprisesa slab housing and a main fluid passage passageway that passes throughthe slab housing. The main fluid passageway is disposed in the slabhousing and can include an inlet, an outlet, and various fluid passagesegments in fluid communication with adjoining fluid passages. The mainfluid passageway can comprise a number of fluid passages collectively influid communication. Such fluid passages can include an initial fluidpassage in fluid communication with the inlet, a transition fluidpassage in fluid communication with the initial fluid passage, mainfluid passage in fluid communication with the transition fluid passage.The main fluid passage can be in direct fluid communication with theoutlet or via an exit fluid passage. The main fluid passage, the initialfluid passage, or both can have a tortuous shape. The main fluidpassage, the initial fluid passage, or both can have a plurality ofcycles (paths) between their respective partitions.

A heating subassembly is also provided by the present teachings and canbe used in combination with one or more thermocycling plates describedherein. The heating subassembly can comprise a first heating block, afirst heat control unit, a second heat control unit, and, optionally, anegative load device. The first heating block can comprise a firststatic heating zone, a heating zone partition, and a second staticheating zone separated from the first static heating zone by the heatingzone partition. The first heat control unit is operably associable withthe first static heating zone. The second heat control unit is operablyassociable with the second static heating zone. The first temperature ortemperature range, or the second temperature or temperature range, canbe a temperature or temperature range sufficient to allow for denaturingof double-stranded nucleic acid, annealing of nucleic acids, orextension of nucleic acids, or any combination thereof. While a singleheating block can be employed in the heating subassembly, use of asecond, complementary heating block can provide additional temperaturecontrol and is particularly advantageous when two thermocycling platesare used. Accordingly, the heating subassembly of the present teachingscan comprise a complementary heating block.

In some embodiments, a method of thermocycling is provided in accordancewith the present teachings. The method can comprise one or more of thefollowing steps, the order of which can be varied, or wherein one ormore of the steps can be repeated. A source solution can be passedthrough a thermocycling plate comprising a plurality of regions. A hotstart region corresponding to an initial fluid passage of thethermocycling plate can be heated and a denaturation regioncorresponding to a portion of a main fluid passage proximal a secondpartition of the thermocycling plate can be heated to a firsttemperature or temperature range. An annealing/extension regioncorresponding to a portion of the main fluid passage proximal a thirdpartition of the thermocycling plate can be heated to a secondtemperature or temperature range. The sample fluid passed through thefluid passageway can comprise a water-in-oil emulsion comprising aplurality of aqueous polymerase chain reaction (PCR) reaction droplets.

In accordance yet other embodiments of the present teachings, acentrifuge subsystem is provided that is suitable for integration intoan emulsion thermocycling system or equivalent device. The centrifugecan comprise a centrifuge housing, a motor comprising a rotor axlemounted in the housing motor aperture, and a rotor mounted on the rotoraxle.

A fluid collection tube is provided that can comprise a main tube bodyand a tube extension. The main tube body can comprise a main bodysidewall surrounding a tube interior with a tube opening at a first end,and a second end distal to the first end providing a sealed base. Thetube extension can comprise a tube extension sidewall defining a fluidexit channel in fluid communication with the tube interior through atube channel inlet proximal to the tube opening and extending to a tubechannel outlet distal to the tube opening.

A fluid distribution device, also referred to herein as a “slinger,” isprovided by the present teachings. The slinger can comprise sidewallsdefining a central channel comprising a first end, a central zone, and asecond end along a longitudinal axis. Sidewall lateral extensions, alsoreferred to as “wings” herein, can be provided extending away from thesidewalls on either side of the central channel. The wings are useful inmating with and ensuring a secure connection with a fluid distributiondevice receptacle of a centrifuge rotor. Rather than use aninsertable/detachable slinger, a slinger can be employed that ispermanently or integrally associated with the rotor housing.

A centrifuge rotor is provided by the present teachings, which isparticularly suitable for separating water-in-oil emulsions and removingthe oil phase of such an emulsion. The centrifuge rotor can comprise arotor housing having a bisecting rotor axis perpendicular to a centralrotor axis, a rotor basin formed by the rotor housing, a rotor basinfloor, and a rotor basin sidewall lining the rotor housing basin andextending up toward a rotor top rim having an inner perimeter and anouter perimeter. The rotor can further comprise at least one collectiontube receptacle comprising an opening formed in the basin sidewall, andat least one tube extension receptacle having a grove formed in therotor top rim and extending from the inner perimeter to the outerperimeter. The rotor can also comprise at least one liquid distributiondevice (slinger) receptacle extending from the rotor basin floor andhaving a distribution device receptacle longitudinal axis.

A method of recovering a polymerase chain reaction (PCR) product from awater-in-oil emulsion is provided in accordance with the presentteachings. The method can be performed using the centrifuge orcentrifuge components of the present teachings. The method can compriseone or more of the following steps. At least one collection tube can befilled with an aqueous solution and inserted into a tube receptacle of acentrifuge rotor. At least one fluid distribution device (slinger) canalso be inserted into a fluid distribution device receptacle of thecentrifuge rotor. Sample fluid, which can comprise a PCR product in awater-in-oil emulsion, can be fed into the fluid distribution device.The centrifuge rotor is spun to deliver the sample fluid to the at leastone collection tube, and the emulsion is partly or completely broken inthe at least one collection tube. The centrifuge rotor motor can also bespun to remove an oil phase from the collection tube. The method canfurther comprise recovering PCR product from the at least one collectiontube.

Taken together it will be appreciated that the disclosed systems andmethods of the present teachings provide an enhanced mechanism by whichto conduct PCR and ePCR reactions using easy to fabricate samplechambers with minimum operator interaction.

It is to be understood that although DNA is referred to often herein,the present teachings also apply to reactions with and emulsionscontaining RNA, PNA, other nucleic acid molecules, other templatemolecules, other reactants, or combinations thereof, instead of or inaddition to DNA.

It is to be understood that each of the publications referenced hereinis independently incorporated herein in its entirety by reference.

In a first aspect, a system for making membrane-based emulsion dropletsincludes an emulsion-generating device comprising a top channel gasket,a bottom channel gasket, an emulsion-generating membrane disposedbetween top channel gasket and bottom channel gasket, a first chamberdefined between top channel gasket and emulsion-generating membrane, anda second chamber defined between emulsion-generating membrane and bottomchannel gasket; a microreactor mixture supply in communication with thetop channel gasket; a carrier fluid supply in communication with thebottom channel gasket; and an emulsion collection device incommunication with the top or bottom channel gasket for collecting themembrane-based emulsion droplets; wherein the emulsion-generatingmembrane comprises at least two through holes.

In an example of the first aspect, each of the membrane-based emulsiondroplets comprises a volume of a microreactor mixture at least partiallysurrounded by the carrier liquid, and the carrier liquid is immisciblewith the microreactor mixture.

In another example of the first aspect and the above examples, the topchannel gasket, the bottom channel gasket, and the emulsion-generatingmembrane are rigidly mounted in a container.

In a further example of the first aspect and the above examples, the topchannel gasket comprises at least one flow path defined therein. Forexample, the at least one flow path is defined in a bottom surface ofthe top channel gasket. In another example, the at least one flow pathcomprises a first flow path and a second flow path.

In an additional example of the first aspect and the above examples, thebottom channel gasket comprises at least one flow path defined therein.For example, the at least one flow path is defined in a top surface ofthe top channel gasket. In another example, the at least one flow pathcomprises a third flow path, a fourth flow path, and a fifth flow path.

In an example of the first aspect and the above examples, at least someof the membrane-based emulsion droplets comprise a microreactor.

In another example of the first aspect and the above examples, themembrane-based emulsion droplets are uniformly sized.

In a further example of the first aspect and the above examples, themicroreactor supply mixture and the carrier fluid supply arepressurized.

In an additional example of the first aspect and the above examples, theemulsion-generating membrane comprises a rubber material.

In an example of the first aspect and the above examples, the firstchamber comprises a volume of microreactor mixture.

In another example of the first aspect and the above examples, the firstchamber comprises a volume of microreactor mixture and at least oneemulsion droplet.

In a further example of the first aspect and the above examples, thesecond chamber comprises a volume of carrier liquid.

In an additional example of the first aspect and the above examples, thesecond chamber comprises a volume of carrier liquid and at least oneemulsion droplet.

In an example of the first aspect and the above examples, the topchannel gasket comprises a first gasket port defined therethrough.

In another example of the first aspect and the above examples, thebottom channel gasket comprises a second gasket port and a third gasketport defined therethrough.

In a second aspect, a method of making membrane-based emulsion dropletsincludes mixing together an aqueous phase solution, a plurality oftemplate beads, a library of templates from a sample, DNA polymerase,and a pair of primers, to form a microreactor mixture; forcing themicroreactor mixture through at least two through holes of anemulsion-generating membrane in an emulsion-generating device, from afirst chamber of the emulsion-generating device and into a carrier fluiddisposed in a second chamber of the emulsion-generating device; forminga plurality of membrane-based emulsion droplets after the microreactormixture passes through the at least two through holes of theemulsion-generating membrane from the first chamber of theemulsion-generating membrane and into the carrier fluid in the secondchamber of the emulsion-generating membrane; and forcing the pluralityof emulsion droplets in the second chamber through the at least twothrough holes of the emulsion-generating membrane into the firstchamber.

In an example of the second aspect, each of the membrane-based emulsiondroplets comprises a volume of a microreactor mixture at least partiallysurrounded by the carrier liquid, and the carrier liquid is immisciblewith the microreactor mixture.

In another example of the second aspect and the above examples, themethod further includes flowing the membrane-based emulsion droplets inat least one flow-path in the first chamber.

In a further example of the second aspect and the above examples, themethod further includes flowing the membrane-based emulsion droplets inat least one flow-path in the second chamber.

In an additional example of the second aspect and the above examples, atleast some of the membrane-based emulsion droplets comprise amicroreactor having at least one template bead.

In an example of the second aspect and the above examples, the methodfurther includes forcing the membrane-based emulsion droplets back andforth through the one or more through holes of the emulsion-generatingmembrane from the first and second chambers.

In another example of the second aspect and the above examples, themembrane-based emulsion droplets are uniformly sized.

In a third aspect, a device for making emulsion droplets includes asubstrate comprising an emulsion-generating plate; a cover in contactwith a top surface of the emulsion-generating plate; a flow cell definedbetween the emulsion-generating plate and the cover; at least twothrough holes extending through at least a portion of theemulsion-generating plate and in fluid communication with the flow cell,the at least two through holes being arranged in a line; a carrier fluidinput port formed through the cover and in fluid communication with theflow cell; and an inverse emulsion outlet port formed through the coverand in fluid communication with the flow cell; wherein the line isarranged at least substantially perpendicularly with respect to adirection from the carrier fluid input port toward the inverse emulsionoutlet port.

In an example of the third aspect, the device further includes a flowcell wall extending between a bottom of the flow cell to the cover.

In another example of the third aspect and the above examples, thedevice further includes a volume of carrier fluid in the flow cell, thecarrier fluid comprising a non-aqueous liquid partially or completelyimmiscible in water.

In a further example of the third aspect and the above examples, thedevice further includes a cavity formed in or below theemulsion-generating plate, the cavity comprising a volume ofmicroreactor mixture and being in fluid communication with the at leasttwo through holes.

In an additional example of the third aspect and the above examples, themicroreactor mixture comprises an aqueous phase solution, a plurality oftemplate beads, a library of templates from a sample, DNA polymerase,and a pair of primers.

In an example of the third aspect and the above examples, the cavity isformed in the emulsion-generating plate.

In another example of the third aspect and the above examples, the atleast two through holes comprises from about 50 to about 100 throughholes.

In a further example of the third aspect and the above examples, the atleast two through holes each has an inner diameter of from about 3microns to about 15 microns.

In an additional example of the third aspect and the above examples, theflow cell comprises nozzles, one around each of the at least two throughholes. For example, the at least two through holes comprises from about50 to about 100 through holes. In another example, each nozzle comprisesan inner diameter of from about 11 microns to about 18 microns.

In a fourth aspect, a method of making an inverse emulsion using thedevice of the first aspect and examples relating thereto includesflowing a pressurized volume of a microreactor mixture into the cavity,the microreactor mixture comprising an aqueous phase solution, aplurality of template beads, a library of templates from a sample, DNApolymerase, and a pair of primers; flowing a pressurized volume of acarrier fluid into the flow cell of the device; forcing the pressurizedvolume of microreactor mixture through the at least two through holesand into the carrier fluid in the flow cell; and forming a plurality ofdroplets of the microreactor mixture as the mixture passes through theat least two through holes and into the carrier fluid in the flow cell,thus forming an inverse emulsion.

In an example of the fourth aspect, each of the emulsion dropletscomprises a volume of a microreactor mixture at least partiallysurrounded by the carrier liquid, and the carrier liquid is immisciblewith the microreactor mixture.

In another example of the fourth aspect and the above examples, themethod further includes forcing the inverse emulsion out of the devicethrough the emulsion outlet port.

In a further example of the fourth aspect and the above examples, theforcing the pressurized volume of the microreactor mixture through theat least two through holes comprises forcing the microreactor mixture toflow in a direction that is perpendicular to the top surface of theemulsion-generating plate.

In an additional example of the fourth aspect and the above examples,the method further includes forcing the microreactor mixture throughnozzles, one nozzle around each of the at least two through holes. Forexample, the flowing the pressurized volume of carrier fluid into theflow cell comprises forcing the pressurized volume of carrier fluid toflow in a direction that is perpendicular to the nozzles around the atleast two through holes.

In an example of the fourth aspect and the above examples, the pluralityof droplets of the microreactor mixture in the inverse emulsion areuniformly sized.

In a fifth aspect, an emulsion-generating device includes (a) a firstchannel plate, said first channel plate comprising (a1) a first fluidport comprising an orifice passing through said first channel plate in athickness direction, and (a2) at least one first flow channel disposedon a bottom surface of said first channel plate; (b) a second channelplate, said second channel plate comprising (b1) a second fluid portcomprising an orifice passing though said second channel plate in athickness direction, and (b2) at least one second flow channel disposedon a top surface of said second channel plate; and (c) a first filtercomprising a plurality of pores, wherein said first filter is disposedbetween said first channel plate and said second channel plate such thatsaid emulsion-generating device comprises a first chamber comprisingsaid bottom surface of said first channel plate and said first filterand a second chamber comprising said top surface of said second channelplate and said first filter.

In an example of the fifth aspect, said at least one first flow channelcomprises two or more flow channels. For example, said two or more flowchannels do not cross each other. In an example, at least two of saidtwo or more flow channels cross each other.

In another example of the fifth aspect and the above examples, said atleast one first flow channel comprises one or more flow channels notconnecting to said first fluid port. For example, said two or more flowchannels do not cross each other. In another example, at least two ofsaid two or more flow channels cross each other.

In a further example of the fifth aspect and the above examples, said atleast one second flow channel comprises a flow channel connecting tosaid second fluid port.

In an additional example of the fifth aspect and the above examples,said at least one second flow channel comprises one or more flowchannels not connecting to said second fluid port.

In an example of the fifth aspect and the above examples, one of saidfirst channel plate and said second channel plate further comprises athird fluid port comprising an orifice passing through said first orsecond channel plate in a thickness direction. For example, said atleast one first or second flow channel comprises a flow channelconnecting to said third fluid port. In an example, said at least onefirst and second flow channel comprises one or more flow channels notconnecting to said third fluid port.

In another example of the fifth aspect and the above examples, thedevice further includes one or more second filters disposed between saidfirst channel plate and said first filter or disposed between saidsecond channel plate and said first filter, each said second filtercomprising a plurality of pores.

In an additional example of the fifth aspect and the above examples, thedevice further includes one or more second filters disposed between saidfirst channel plate and said first filter and one or more third filtersdisposed between said second channel plate and said first filter, saidsecond and third filter comprising a plurality of pores.

In an example of the fifth aspect and the above examples, said pluralityof pores have a size of about 1 to about 50 microns.

In another example of the fifth aspect and the above examples, saidfirst filter is a membrane.

In a further example of the fifth aspect and the above examples, saidfirst filter is a track-etched filter.

In an additional example of the fifth aspect and the above examples,said first filter is a laser-etched filter.

In an example of the fifth aspect and the above examples, the devicefurther includes a housing, wherein said first channel plate, saidsecond channel plate, and said first filter are mounted in said housing.

In another example of the fifth aspect and the above examples, said atleast one first flow channel or said at least one second flow channel isconfigured to have low fluid resistance.

In a further example of the fifth aspect and the above examples, said atleast one first flow channel or said at least one second flow channel isdisposed such that fluid passes said first filter a plurality of times.

In an additional example of the fifth aspect and the above examples,said at least one first flow channel or said at least one second flowchannel has a depth of not greater than 500 μm, and wherein said firstchamber or said second chamber has a depth from said first or secondchannel plate to said first filter of not greater than 500 μm.

In a sixth aspect, a system for making emulsion droplets includes anemulsion-generating device comprising: (a) a first channel plate, saidfirst channel plate comprising (a1) a first fluid port comprising anorifice passing through said first channel plate in a thicknessdirection, and (a2) at least one first flow channel disposed on a bottomsurface of said first channel plate; (b) a second channel plate, saidsecond channel plate comprising (b1) a second fluid port comprising anorifice passing though said second channel plate in a thicknessdirection, and (b2) at least one second flow channel disposed on a topsurface of said second channel plate; and (c) a first filter comprisinga plurality of pores, wherein said first filter is disposed between saidfirst channel plate and said second channel plate such that saidemulsion-generating device comprises a first chamber comprising saidbottom surface of said first channel plate and said first filter and asecond chamber comprising said top surface of said second channel plateand said first filter; a reaction mixture supply in fluid communicationwith said first chamber by way of said first fluid port; a carrier fluidsupply in fluid communication with said second chamber by way of saidsecond fluid port; and an emulsion collection device in fluidcommunication with said second chamber by way of said third fluid port.

In an example of the sixth aspect, said reaction mixture supply and saidcarrier fluid supply are pressurized.

In a seventh aspect, a method of making emulsion droplets in anemulsion-generating device includes (i) a first channel plate, saidfirst channel plate comprising (i1) a first fluid port comprising anorifice passing through said first channel plate in a thicknessdirection, and (i2) at least one first flow channel disposed on a bottomsurface of said first channel plate; (ii) a second channel plate, saidsecond channel plate comprising (ii1) a second fluid port comprising anorifice passing though said second channel plate in a thicknessdirection, and (ii2) at least one second flow channel disposed on a topsurface of said second channel plate; and (iii) a first filtercomprising a plurality of pores, wherein said first filter is disposedbetween said first channel plate and said second channel plate such thatsaid emulsion-generating device comprises a first chamber comprisingsaid bottom surface of said first channel plate and said first filterand a second chamber comprising said top surface of said second channelplate and said first filter; said method comprising: (a) flowing areaction mixture into said first fluid port of said emulsion-generatingdevice; (b) flowing a carrier fluid into said second fluid port of saidemulsion-generating device, wherein said carrier fluid is immisciblewith said reaction mixture; and (c) recovering an emulsion fluid fromsaid third fluid port, wherein said emulsion fluid comprises droplets ofsaid reaction mixture in said carrier fluid.

In an example of the seventh aspect, said reaction mixture comprises aplurality of template beads, a plurality of templates, a DNA polymerase,and a pair of primers

In another example of the seventh aspect and the above examples, themethod further includes adjusting a fluid pressure of said reactionmixture or adjusting a fluid pressure of said carrier fluid such thatsaid droplets of said reaction mixture in said carrier fluid in saidstep (c) have a predetermined size.

In an eighth aspect, a method of making emulsion droplets includes (a)passing a flow of reaction mixture and carrier fluid such that saidreaction mixture passes through a filter from a first side to a secondside and said carrier fluid flows, said filter comprising a plurality ofpores, (b) creating a shear flow of a carrier fluid at said second sideto generate a first emulsion comprising a plurality of first droplets ofsaid reaction mixture in said carrier fluid, wherein said carrier fluidis immiscible with said reaction mixture; (c) passing a flow of saidfirst emulsion through said filter from said second side to said firstside; (d) creating a shear flow by said carrier fluid at said first sideto generate a second emulsion comprising a plurality of second dropletsof said reaction mixture in said carrier fluid; and (e) recovering saidsecond emulsion fluid.

In a ninth aspect, am emulsion-generating device includes a first plateand a second plate configured to form a flow chamber; a fluid input portand a fluid output port in fluid communication with said flow chamber;at least said first or second plate comprising a plurality of throughholes passing through said first or second plate in a plate thicknessdirection; said plurality of through holes being disposed in one or morelines oriented at a substantially perpendicular direction with respectto a direction from said fluid input port toward said fluid outlet port.

In an example of the ninth aspect, said first plate is a top plate andsaid second plate is a bottom plate, and wherein said bottom platecomprises said plurality of through holes.

In another example of the ninth aspect and the above examples, saidplurality of through holes comprises from about 50 to about 100 throughholes. For example, said plurality of through holes each has an innerdiameter of from about 3 microns to about 15 microns.

In a further example of the ninth aspect and the above examples, thedevice further includes a respective elevated rim around each of saidthrough hole.

In an additional example of the ninth aspect and the above examples,each said elevated rim has an inner diameter from about 11 microns toabout 18 microns.

In a tenth aspect, a system for making emulsion droplets includes theemulsion-generating device of the ninth aspect and its associatedexamples; a reaction mixture supply in fluid communication with saidflow chamber by way of said plurality of through holes; a carrier fluidsupply in fluid communication with said flow chamber by way of saidfluid inlet port; and an emulsion collection device in fluidcommunication with said flow chamber by way of said outlet fluid port.

In an example of the tenth aspect, said reaction mixture supply and saidcarrier fluid supply are pressurized.

In another example of the tenth aspect and the above example, saidreaction mixture supply includes a sample tube.

In an eleventh aspect, a method of making an inverse emulsion includesproviding a reaction mixture, a carrier fluid, and a device of the ninthaspect and associated examples, wherein said carrier fluid is immisciblewith said reaction mixture; flowing a reaction mixture into said flowchamber through said through holes; flowing a carrier fluid into saidflow chamber through said fluid inlet; and forming a plurality ofdroplets of said reaction mixture in said carrier fluid, thus forming aninverse emulsion.

In an example of the eleventh aspect, the reaction mixture comprises anaqueous phase solution, a plurality of template beads, a library oftemplates from a sample, DNA polymerase, and a pair of primers.

In another example of the eleventh aspect and the above examples, themethod further includes recovering said inverse emulsion from said fluidoutlet port.

In a further example of the eleventh aspect and the above examples, themethod further includes adjusting a fluid pressure of said reactionmixture or adjusting a fluid pressure of said carrier fluid such thatsaid droplets of said reaction mixture in said carrier fluid have apredetermined size.

In a twelfth aspect, a sample reaction plate includes a slab housinghaving a width, a length, and a thickness less than both the width andthe length, and comprising a plurality of corners comprising a firstcorner, a second corner, a third corner, and a fourth corner, aplurality of edges comprising a first edge extending from the firstcorner to the second corner, a second edge extending from the secondcorner to the third corner, a third edge extending from the third cornerto the fourth corner, and a fourth edge extending from the fourth cornerto the first corner; a plurality of partitions extending across thewidth and comprising a first partition proximal the first edge, a secondpartition between the first partition and a third partition, and thethird partition proximal the third edge; and a fluid passageway disposedin the housing, said fluid passageway comprising an inlet proximal thefirst corner, an initial fluid passage in fluid communication with theinlet and extending to proximal the second corner along the width of theslab housing, between the first partition and the second partition, atransition fluid passage in fluid communication with the initial fluidpassage and extending from proximal the second corner to proximal thethird corner along the length of the slab housing, a main fluid passagein fluid communication with the transition fluid passage, extending fromproximal the third corner to proximal the fourth corner along the widthof the slab housing, comprising a plurality of repeats between thesecond partition and the third partition, and having a tortuous shape,and an outlet in fluid communication with the main fluid passage.

In an example of the twelfth aspect, the second partition is parallel toat least one of the first partition and the third partition.

In another example of the twelfth aspect and the above examples, thedistance between the second and third partitions is greater than thedistance between the first and second partitions.

In a further example of the twelfth aspect and the above examples, thedistance between the second and third partitions is at least five timesgreater than the distance between the first and second partitions.

In an additional example of the twelfth aspect and the above examples,the distance between the second and third partitions is at least tentimes greater than the distance between the first and second partitions.

In an example of the twelfth aspect and the above examples, the initialfluid passage has a tortuous shape and comprises a plurality of repeatsbetween the first partition and the second partition.

In another example of the twelfth aspect and the above examples, theinitial fluid passage has a tortuous shape and comprises: a plurality ofrepeats between the first partition and the second partition formed by aplurality of initial straight members, and a plurality of initial turnmembers joining the initial straight members at the first and secondpartitions; and the main fluid passage comprising said plurality ofrepeats between the second partition and the third partition formed by aplurality of main straight members, and a plurality of main turn membersjoining the main straight members at the second and third partitions.For example, the main straight members are longer than the initialstraight members. In an example, the main straight members are at leastfive times longer than the initial straight members. In another example,the main straight members are at least ten times longer than the initialstraight members.

In a further example of the twelfth aspect and the above examples, themain fluid passage comprises from about 5 to about 500 repeats betweenthe second partition and the third partition.

In an additional example of the twelfth aspect and the above examples,the main fluid passage comprises from about 10 to about 100 repeatsbetween the second partition and the third partition.

In an example of the twelfth aspect and the above examples, the initialfluid passage comprises from about 10 repeats to about 100 repeatsbetween the first partition and the second partition.

In another example of the twelfth aspect and the above examples, themethod further includes an exit fluid passage in fluid communicationwith the main fluid passage and extending from proximal the fourthcorner to proximal the first corner along the length of the slabhousing, and wherein the outlet is proximal the first corner in fluidcommunication with the exit fluid passage.

In a further example of the twelfth aspect and the above examples, atleast one initial turn member or main turn member has an averagecross-section area less than an average cross-section area of anadjacent initial straight member or adjacent main straight member. Forexample, a plurality of the initial turn members along the secondpartition each have an average cross-sectional area less than theaverage cross-sectional area of an adjacent initial straight member. Inanother example, a plurality of the main turn members along the thirdpartition each have an average cross-sectional area less than theaverage cross-sectional area of an adjacent initial straight member. Ina further example, a plurality of the initial turn members along thefirst partition each have an average cross-sectional area less than theaverage cross-sectional area of an adjacent initial straight member. Inan additional example, a plurality of the main turn members along thesecond partition each have an average cross-sectional area less than theaverage cross-sectional area of an adjacent initial straight member.

In an additional example of the twelfth aspect and the above examples,the plate further includes a first face bounded by the plurality ofedges and plurality of corners; and a second face parallel to the firstface; bounded by the plurality of edges and plurality of corners;wherein the inlet and outlet are comprised by the first face or thesecond face.

In a thirteenth aspect, a pair of thermocycling plates includes a firstthermocycling plate corresponding to the thermocycling plate of thetwelfth aspect and associated examples; and a second thermocycling platecomprising a mirror-image configuration of the thermocycling plate.

In a fourteenth aspect, a heating subassembly includes a first heatingblock comprising a first static heating zone, a heating zone partition,and a second static heating zone separated from the first static heatingzone by the heating zone partition; a first heat control unit operablyassociated with the first static heating zone; a second heat controlunit operably associated with the second static heating zone; and apower source electrically associated with the first and second heatcontrol units.

In an example of the fourteenth aspect, the subassembly further includesa negative load device operably associated with the second heat controlunit and the second static heating zone. For example, the first heatcontrol unit is configured to maintain the first static heating zone ata first temperature or within in a first temperature range; the secondheat control unit is configured to maintain the first static heatingzone at a second temperature or within in a second temperature range;and the first temperature is higher than the second temperature, and thefirst temperature range is higher than second temperature range. Inanother example, the first temperature range does not overlap with thesecond temperature range. In a particular example, the first temperatureor temperature range differs by at least 10° C. from the secondtemperature or temperature range. In another example, the firsttemperature range is from about 85° C. to about 100° C.; and the secondtemperature range is from about 45° C. to about 75° C.

In another example of the fourteenth aspect and the above examples, thenegative load device comprises a fan.

In a further example of the fourteenth aspect and the above examples,the subassembly further includes a recess configured to allow passage ofa sample reaction plate inlet, a sample reaction plate outlet, or tubingassociated with at least one of the inlet and the outlet, or anycombination thereof.

In an additional example of the fourteenth aspect and the aboveexamples, the subassembly further includes a complementary heating blockcomprising: a first complementary static heating zone; a complementaryheating zone partition; and a second complementary static heating zoneseparated from the first complementary static heating zone by thecomplementary heating zone partition. For example, the complementaryheating block comprises: a first complementary heat control unitoperably associated with the first complementary static heating zone; asecond complementary heat control unit operably associated with thesecond complementary static heating zone; and a complementary negativeload device operably associated with the second complementary heatcontrol unit and the second complementary static heating zone. In anexample, the second heating block is electrically associated with thesecond first and second complementary heat control units.

In a fifteenth aspect, an amplification system includes a first samplereaction plate comprising a slab housing having a width, a length, and athickness less than both the width and the length, and comprising aplurality of corners comprising a first corner, a second corner, a thirdcorner, and a fourth corner, a plurality of edges comprising a firstedge extending from the first corner to the second corner, a second edgeextending from the second corner to the third corner, a third edgeextending from the third corner to the fourth corner, and a fourth edgeextending from the fourth corner to the first corner; a plurality ofpartitions extending across the width and comprising a first partitionproximal the first edge, a second partition between the first partitionand a third partition, and the third partition proximal the third edge,and a fluid passageway disposed in the housing and comprising an inletproximal the first corner, an initial fluid passage in fluidcommunication with the inlet and extending to proximal the second corneralong the width of the slab housing, between the first partition and thesecond partition, a transition fluid passage in fluid communication withthe initial fluid passage and extending from proximal the second cornerto proximal the third corner along the length of the slab housing, amain fluid passage in fluid communication with the transition fluidpassage, extending from proximal the third corner to proximal the fourthcorner along the width of the slab housing, comprising a plurality ofrepeats between the second partition and the third partition, and havinga tortuous shape, and an outlet in fluid communication with the mainfluid passage; and a heating subassembly comprising a first heatingblock comprising a first static heating zone, a heating zone partition,and a second static heating zone separated from the first static heatingzone by the heating zone partition, a first heat control unit operablyassociated with the first static heating zone, a second heat controlunit operably associated with the second static heating zone, whereinthe first sample reaction plate and first heating block are adjacent,parallel, and in thermal communication with each other, with the firststatic heating zone in alignment with a hot start region correspondingto the initial fluid passage and a denaturation region corresponding toa portion of the main fluid passage proximal the second partition, andthe second static heating zone in alignment with an annealing/extensionregion corresponding to a portion of the main fluid passage proximal thethird partition.

In an example of the fifteenth aspect, the system further includes anegative load device operably associated with the second heat controlunit and the second static heating zone.

In another example of the fifteenth aspect and the above examples, thesystem further includes a complementary heating block comprising: afirst complementary static heating zone; a complementary heating zonepartition; and a second complementary static heating zone separated fromthe first complementary static heating zone by the complementary heatingzone partition; wherein the first sample reaction plate andcomplementary heating block are adjacent, parallel, and in thermalcommunication with each other, the first complementary static heatingzone in alignment with a hot start region corresponding to the initialfluid passage and a denaturation region corresponding to a portion ofthe main fluid passage proximal the second partition, and the secondcomplementary static heating zone in alignment with anannealing/extension region corresponding to a portion of the main fluidpassage proximal the third partition.

In another example of the fifteenth aspect and the above examples, thesystem further includes a second sample reaction plate comprising thesame characteristics of the first sample reaction plate; and acomplementary heating block comprising a first complementary staticheating zone, a complementary heating zone partition, and a secondcomplementary static heating zone separated from the first complementarystatic heating zone by the complementary heating zone partition; whereinthe first and second sample reaction plates are adjacent to one another,and the second sample reaction plate and complementary heating block areadjacent, parallel, and in thermal communication with each other, thefirst complementary static heating zone in alignment with a hot startregion corresponding to the initial fluid passage of the second samplereaction plate and a denaturation region corresponding to a portion ofthe main fluid passage proximal the second partition of the secondsample reaction plate, and the second complementary static heating zonein alignment with an annealing/extension region corresponding to aportion of the main fluid passage proximal the third partition of thesecond sample reaction plate.

In a further example of the fifteenth aspect and the above examples, thesystem further includes a gasket adjacent to at least three sides ofeach of the first sample reaction plate and the first heating block.

In an additional example of the fifteenth aspect and the above examples,the system further includes a gasket adjacent to at least three sides ofeach of the first sample reaction plate, the second sample reactionplate, the first heating block, and the complementary heating block.

In a sixteenth example, a method of thermocycling includes passing asample fluid through a sample reaction plate comprising a plurality ofregions; heating a hot start region corresponding to an initial fluidpassage of the sample reaction plate and heating a denaturation regioncorresponding to a portion of a main fluid passage proximal a secondpartition of the sample reaction plate to a first temperature ortemperature range; heating an annealing/extension region correspondingto a portion of the main fluid passage proximal a third partition of thesample reaction plate to a second temperature or temperature range;wherein the first temperature is higher than the second temperature, andthe first temperature range is higher than second temperature range, andwherein the sample reaction plate comprises a slab housing having awidth, a length, and a thickness less than both the width and thelength, and comprising a plurality of corners comprising a first corner,a second corner, a third corner, and a fourth corner, a plurality ofedges comprising a first edge extending from the first corner to thesecond corner, a second edge extending from the second corner to thethird corner, a third edge extending from the third corner to the fourthcorner, and a fourth edge extending from the fourth corner to the firstcorner, a plurality of partitions extending across the width andcomprising a first partition proximal the first edge, a second partitionbetween the first partition and a third partition, and the thirdpartition proximal the third edge; and a fluid passageway disposed inthe housing and comprising an inlet proximal the first corner, aninitial fluid passage in fluid communication with the inlet andextending to proximal the second corner along the width of the slabhousing, between the first partition and the second partition, atransition fluid passage in fluid communication with the initial fluidpassage and extending from proximal the second corner to proximal thethird corner along the length of the slab housing, a main fluid passagein fluid communication with the transition fluid passage, extending fromproximal the third corner to proximal the fourth corner along the widthof the slab housing, comprising a plurality of repeats between thesecond partition and the third partition, and having a tortuous shape,and an outlet in fluid communication with the main fluid passage whereinsaid passing said fluid through said sample reaction plate comprisesfeeding said fluid into said inlet.

In an example of the sixteenth aspect, the sample fluid comprises awater-in-oil emulsion comprising a plurality of aqueous polymerase chainreaction (PCR) reaction droplets.

In another example of the sixteenth aspect and the above examples, themethod further includes annealing a sample nucleic acid in the reactordroplet to a template in the reactor droplet; extending the samplenucleic acid to form a double-stranded nucleic acid; and denaturing thedouble-stranded nucleic acid.

In an additional example of the sixteenth aspect and the above examples,the method further includes sending PCR product from the outlet to acentrifuge for recovery of the PCR product.

In a seventeenth aspect, a fluid collection tube includes a main tubebody comprising a main body housing surrounding a tube interiorcomprising a tube opening at a first end, and a second end distal to thefirst end providing a sealed base; and a tube extension comprising atube extension sidewall defining a fluid exit channel in fluidcommunication with the tube interior through a tube channel inletproximal to the tube opening and extending to a tube channel outletdistal to the tube opening.

In an example of the seventeenth aspect, the tube further includes atube lip disposed about the perimeter of the tube opening and allowingfluid communication of the tube exit channel with the tube interior.

In another example of the seventeenth aspect and the above examples, thetube further includes a tube buttress disposed between the tubeextension sidewall and the main tube housing.

In an additional example of the seventeenth aspect and the aboveexamples, the tube extension sidewall and the main tube housing arepositioned at an angle of from about 15° to about 90° to each other.

In a further example of the seventeenth aspect and the above examples,the tube extension sidewall and the main tube housing are positioned atan angle of from about 30° to about 60° relative to each other.

In an example of the seventeenth aspect and the above examples, the maintube body comprises a generally cylindrical portion proximal the firstend, a conically tapered portion proximal the second end, and a roundedsecond end.

In another example of the seventeenth aspect and the above examples, thetube exit channel and tube extension sidewall have a U-shapedcross-section along a longitudinal axis.

In a further example of the seventeenth aspect and the above examples,an average cross-sectional area of the exit tube channel is less than anaverage cross-sectional area of the tube interior.

In an additional example of the seventeenth aspect and the aboveexamples, the average cross-sectional area of the exit tube channel isless than about 25% of the average cross-sectional area of the tubeinterior.

In an example of the seventeenth aspect and the above examples, the tubefurther includes a cap.

In an eighteenth aspect, a fluid distribution device includes sidewallsdefining a central channel comprising a first end, a central zone, and asecond end along a longitudinal axis; wings extending away from thesidewalls on either side of the central channel; a first spout extendingfrom the central zone to the first end and terminating at a first spoutoutlet, the sidewalls tapering along the central zone toward the firstspout; and a second spout extending from the central zone to the secondend and terminating at a second spout outlet, the sidewalls taperingalong the central zone toward the second spout.

In an example of the eighteenth aspect, the device is configured to matewith a fluid distribution device receptacle of a centrifuge rotor.

In a nineteenth aspect, a centrifuge rotor includes a rotor housinghaving a bisecting rotor axis perpendicular to a central rotor axis; arotor basin formed by the rotor housing; a rotor basin floor; a rotorbasin sidewall lining the rotor housing basin and extending up toward arotor top rim having an inner perimeter and an outer perimeter; at leastone collection tube receptacle comprising an opening formed in the basinsidewall; at least one tube extension receptacle comprising a groveformed in the rotor top rim and extending from the inner perimeter tothe outer perimeter; and at least one liquid distribution devicereceptacle extending from the rotor basin floor and having adistribution device receptacle longitudinal axis.

In an example of the nineteenth aspect, the collection tube receptacleopening and the tube extension groove are positioned at an angle of fromabout 15° to about 90° relative to each other.

In another example of the nineteenth aspect and the above examples, thecollection tube receptacle opening and the tube extension groove arepositioned at an angle of from about 30° to about 60° relative to eachother.

In a further example of the nineteenth aspect and the above examples,the fluid distribution device receptacle comprises opposing sidewalls oneither side of the distribution device receptacle longitudinal axis.

In an additional example of the nineteenth aspect and the aboveexamples, the bisecting rotor axis is not parallel to the distributiondevice receptacle longitudinal axis. For example, the axes arepositioned from about 0.15° to about 15.0° relative to each other. Inanother example, the axes are positioned from about 0.5° to about 5.0°relative to each other.

In an example of the nineteenth aspect and the above examples, thesidewalls comprise a substantially flat inset region about thecollection tube receptacle and adjacent the tube receptacle opening.

In another example of the nineteenth aspect and the above examples, therotor includes first and second tube receptacles positioned oppositeeach other along the bisecting rotor axis; and first and second tubeextension grooves opposite each other along the bisecting rotor axis.

In a twentieth aspect, a centrifuge includes a centrifuge housingcomprising centrifuge housing sidewalls extending from a base portion toa top portion comprising a top housing rim surrounding a top housingaperture, a lid operably associable with the top housing rim, a housingbottom recess extending from the base to an interior of the centrifugehousing and comprising bottom recess sidewalls and a bottom recessceiling, a housing basin defined by the top housing rim, the top housingaperture, and housing basin sidewalls surrounding a receiving platform,and a housing motor aperture configured to accept a motor through thebottom recess ceiling and the receiving platform; a motor comprising arotor axle and mounted in the housing motor aperture; and a rotormounted on the rotor axle and comprising a rotor housing having abisecting rotor axis perpendicular to a central rotor axis; a rotorbasin formed by the rotor housing; a rotor basin floor; a rotor basinsidewall lining the rotor housing basin and extending up from the rotorbasin floor toward a rotor top rim having an inner perimeter and anouter perimeter; at least one collection tube receptacle comprising anopening formed in the basin sidewall; at least one tube extensionreceptacle having a grove formed in the rotor top rim and extending fromthe inner perimeter to the outer perimeter; and at least one liquiddistribution device (slinger) receptacle extending from the rotor basinfloor and having a distribution device receptacle longitudinal axis.

In an example of the twentieth aspect, the centrifuge further includes ahousing hinge assembly including a housing hinge plate operativelyconnectable to the centrifuge housing and comprising a top plateportion; first and second hinge receiving arms extending from the topplate portion and comprising respective first and second hinge axleapertures; and a hinge axle extending from the first and second hingeaxle apertures and passing through a lid hinge portion extending fromthe lid.

In another example of the twentieth aspect and the above examples, thelid further comprises a top lid surface and a bottom lid surface; acentral lid aperture extending from the top lid surface to the bottomlid surface; wherein the central lid aperture is configured to accept afluid supply line and positioned above the fluid distribution devicewhen the lid is in a closed configuration. For example, the centrifugefurther includes a fluid supply source in fluid communication with thefluid supply line; and a fluid supply pump configured to pump fluid fromthe fluid supply source, through the fluid supply line and into thecentrifuge.

In a further example of the twentieth aspect and the above examples, thecentrifuge further includes a peripheral gutter comprising: a gutterhousing providing a top gutter surface, a bottom gutter surface, andgutter sidewalls extending between the top and bottom gutter surfacesalong an outer gutter perimeter of the gutter housing; a gutter inletalong an inner perimeter of the gutter housing; and a gutter flangeextending around the gutter outer perimeter and adapted for placement onthe top housing rim. For example, the centrifuge further includes agutter outlet in the gutter housing; a housing drainage aperture in thehousing sidewall; a basin drainage aperture in the housing basinsidewall; and drainage tubing operatively associated with the gutteroutlet and passing through the drainage apertures.

In a twenty-first aspect, a method of recovering a polymerase chainreaction (PCR) product from a water-in-oil emulsion includes filling atleast one collection tube with an aqueous solution; inserting the atleast one collection tube into a tube receptacle of a centrifuge rotor;inserting at least one fluid distribution device (slinger) into a fluiddistribution device receptacle of the centrifuge rotor; feeding samplefluid comprising an emulsion into the fluid distribution device;spinning the centrifuge rotor to deliver the sample fluid to the atleast one collection tube; breaking the emulsion in the at least onecollection tube; and spinning the centrifuge rotor to remove an oilphase from the collection tube.

In an example of the twenty-first aspect, the emulsion comprises awater-in-oil emulsion

In another example of the twenty-first aspect and the above examples,the aqueous solution comprises at least one of a surfactant and adetergent. For example, the aqueous solution comprises sodium dodecylsulfate. In another example, the aqueous solution further comprisesethanol.

In a further example of the twenty-first aspect and the above examples,the at least one collection tube comprises at least one mixing ballcapable of capturing PCR product associated beads.

In an additional example of the twenty-first aspect and the aboveexamples, the method further includes collecting the oil phase in aperipheral gutter.

In an example of the twenty-first aspect and the above examples, thesample fluid comprises a PCR product and further comprising recoveringPCR product from the at least one collection tube.

In another example of the twenty-first aspect and the above examples,the fluid distribution device is inserted such that at least one fluiddistribution outlet is displaced off center from a center of the atleast one collection tube; and the spinning occurs in the direction ofthe displacement. For example, a degree of the displacement is relativeto at least one of an angular velocity and an angular acceleration ofthe spinning rotor. In an example, the degree of the displacementresults in sample fluid entering the at least one collection tube at ornear a center of the tube.

In a further example of the twenty-first aspect and the above examples,the method further includes mixing the contents of the at least onecollection tube by alternating the direction of the spinning rotor.

In an twenty-second aspect, a centrifuge includes a rotor comprising arotor housing having a bisecting rotor axis perpendicular to a centralrotor axis; a rotor basin formed by the rotor housing; a rotor basinfloor; a rotor basin sidewall lining the rotor housing basin andextending up from the rotor basin floor toward a rotor top rim having aninner perimeter and an outer perimeter; at least one collection tubereceptacle comprising an opening formed in the basin sidewall; at leastone tube extension receptacle having a grove formed in the rotor top rimand extending from the inner perimeter to the outer perimeter; and atleast one liquid distribution device (slinger) receptacle extending fromthe rotor basin floor and having a distribution device receptaclelongitudinal axis.

In an example of the twenty-second aspect, the centrifuge furtherincludes a centrifuge housing comprising centrifuge housing sidewallsextending from a base portion to a top portion comprising a top housingrim surrounding a top housing aperture, a lid operably associable withthe top housing rim, a housing bottom recess extending from the base toan interior of the centrifuge housing and comprising bottom recesssidewalls and a bottom recess ceiling, a housing basin defined by thetop housing rim, the top housing aperture, and housing basin sidewallssurrounding a receiving platform, and a housing motor apertureconfigured to accept a motor through the bottom recess ceiling and thereceiving platform; a motor comprising a rotor axle and mounted in thehousing motor aperture; wherein said rotor is mounted on the rotor axle.For example, the centrifuge further includes a housing hinge assemblycomprising: a housing hinge plate operatively connectable to thecentrifuge housing and comprising a top plate portion; first and secondhinge receiving arms extending from the top plate portion and comprisingrespective first and second hinge axle apertures; and a hinge axleextending from the first and second hinge axle apertures and passingthrough a lid hinge portion extending from the lid.

In another example of the twenty-second aspect and the above examples,the lid further comprises: a top lid surface and a bottom lid surface; acentral lid aperture extending from the top lid surface to the bottomlid surface; wherein the central lid aperture is configured to accept afluid supply line and positioned above the fluid distribution devicewhen the lid is in a closed configuration. For example, the centrifugefurther includes a fluid supply source in fluid communication with thefluid supply line; and a fluid supply pump configured to pump fluid fromthe fluid supply source, through the fluid supply line and into thecentrifuge.

In a further example of the twenty-second aspect and the above examples,the centrifuge further includes a peripheral gutter comprising: a gutterhousing providing a top gutter surface, a bottom gutter surface, andgutter sidewalls extending between the top and bottom gutter surfacesalong an outer gutter perimeter of the gutter housing; a gutter inletalong an inner perimeter of the gutter housing; and a gutter flangeextending around the gutter outer perimeter and adapted for placement onthe top housing rim. For example, the centrifuge further includes agutter outlet in the gutter housing; a housing drainage aperture in thehousing sidewall; a basin drainage aperture in the housing basinsidewall; and drainage tubing operatively associated with the gutteroutlet and passing through the drainage apertures.

In a twenty-third aspect a method of separating a product in an aqueousphase from an inverse emulsion comprising an aqueous phase and a waterimmiscible phase using a centrifuge including a rotor comprising a rotorhousing having a bisecting rotor axis perpendicular to a central rotoraxis; a rotor basin formed by the rotor housing; a rotor basin floor; arotor basin sidewall lining the rotor housing basin and extending upfrom the rotor basin floor toward a rotor top rim having an innerperimeter and an outer perimeter; at least one collection tubereceptacle comprising an opening formed in the basin sidewall, whereinsaid collection tube receptacle containing a collection tube filled withan aqueous solution; at least one tube extension receptacle having agrove formed in the rotor top rim and extending from the inner perimeterto the outer perimeter; and at least one liquid distribution device(slinger) receptacle extending from the rotor basin floor and having adistribution device receptacle longitudinal axis, wherein said liquiddistribution device receptacle containing a liquid distribution device,the method comprising: feeding a sample fluid comprising said inverseemulsion into the fluid distribution device; spinning the centrifugerotor to deliver the sample fluid to the at least one collection tubeand to remove said water immiscible phase from the collection tube,wherein the emulsion is broken in the at least one collection tube.

In an example of the twenty-third aspect, the method further includes,prior to said step of feeding sample fluid, inserting a collection tubeinto said tube receptacle; and inserting a fluid distribution device(slinger) into said fluid distribution device receptacle.

In another example of the twenty-third aspect and the above examples,the inverse emulsion comprises a water-in-oil emulsion.

In a further example of the twenty-third aspect and the above examples,the aqueous solution comprises at least one of a surfactant and adetergent. For example, the aqueous solution comprises sodium dodecylsulfate.

In an additional example of the twenty-third aspect and the aboveexamples, the aqueous solution further comprises ethanol.

In an example of the twenty-third aspect and the above examples, the atleast one collection tube comprises at least one mixing ball capable ofcapturing PCR product associated beads.

In another example of the twenty-third aspect and the above examples,the method further includes collecting the water immiscible phase ofsaid inverse emulsion in a peripheral gutter.

In a further example of the twenty-third aspect and the above examples,the sample fluid comprises a PCR product and said method furthercomprising recovering PCR product from the at least one collection tube.

In an additional example of the twenty-third aspect and the aboveexamples, the fluid distribution device is inserted such that at leastone fluid distribution outlet is displaced off center from a center ofthe at least one collection tube; and the spinning occurs in thedirection of the displacement. For example, a degree of the displacementis relative to at least one of an angular velocity and an angularacceleration of the spinning rotor. In another example, the degree ofthe displacement results in sample fluid entering the at least onecollection tube at or near a center of the tube.

In an example of the twenty-third aspect and the above examples, themethod further includes mixing the contents of the at least onecollection tube by alternating the direction of the spinning rotor.

In another example of the twenty-third aspect and the above examples,the method further includes performing said feeding said sample fluidinto the fluid distribution device and said spinning the centrifugerotor continuously.

In a twenty-fourth aspect, an emulsion generating device includes afirst gasket including a first set of channels; a second gasketincluding a second set of channels complementary to the first set ofchannels, a carrier fluid inlet and an emulsion outlet fluidicallycoupled to the second set of channels; and a membrane disposed betweenthe first and second gaskets, wherein fluid is to pass through themembrane at least three times when traversing the first and second setsof channels between the carrier fluid inlet and the emulsion outlet.

In an example of the twenty-fourth aspect, the first and second sets ofchannels are concurrently complementary.

In another example of the twenty-fourth aspect and the above examples,the first and second set of channels are countercurrently complementary.

In a further example of the twenty-fourth aspect and the above examples,the membrane comprises laser etched holes.

In an additional example of the twenty-fourth aspect and the aboveexamples, the depth of the first or second set of channels is notgreater than 500 micrometers.

In an example of the twenty-fourth aspect and the above examples, thedevice further includes a housing, the first and second channel gasketand the membrane disposed within the housing. For example, the housingdefines a carrier fluid inlet port in fluidic communication with thecarrier fluid inlet of the second gasket and defines an emulsion outletport in fluid communication with the emulsion outlet of the secondgasket. In another example, the housing defines a sample tubereceptacle. In a further example, the housing further defines adisplacement fluid inlet port in fluid communication with the sampletube receptacle. In an additional example, the device further includes asample fluid tube.

In a twenty-fifth aspect, an emulsion generating system includes anemulsion generating device comprising: a housing defining a sample inletport, a carrier fluid inlet port, an emulsion outlet port, and adisplacement fluid inlet port; first and second channel gaskets; and amembrane disposed between the first and second channel gaskets; and asample tube disposed on the sample inlet port and in fluid communicationwith the displacement fluid inlet port.

In an example of the twenty-fifth aspect, the carrier fluid inlet portand the emulsion outlet port are disposed on a lower surface of thehousing.

In another example of the twenty-fifth aspect and the above examples,the sample inlet port is disposed on an upper surface of the housing.For example, the displacement fluid port is disposed on the uppersurface of the housing. In another example, the displacement fluid portis disposed on a lower surface of the housing, a passageway definedbetween the displacement fluid port and the sample tube through thefirst and second channel gaskets and the membrane.

In a further example of the twenty-fifth aspect and the above examples,the first and second channel gaskets define a first set and a second setof complementary channels. For example, the first and second set ofcomplementary channels are concurrently complementary. In anotherexample, the first and second set of complementary channels arecountercurrently complementary. In an additional example, the first andsecond set of complementary channels have a depth of not greater than500 micrometers.

In a twenty-sixth aspect, a method of generating an emulsion includesapplying an aqueous sample solution to the sample tube of the emulsiongenerating system of the twenty-fifth aspect and associated examples,applying a displacement fluid through the displacement fluid inlet port;applying a carrier fluid through the carrier fluid inlet port; andcollecting an emulsion through the emulsion outlet port.

In an example of the twenty-sixth aspect, the displacement fluid and thecarrier fluid comprise an oil.

In another example of the twenty-sixth aspect and the above examples,the displacement fluid displaces the aqueous sample fluid from thesample tube.

In a twenty-eighth aspect, a thermocycling device includes a platecomprising an inlet port and an outlet port and defining a first channelon a first side of the plate and a second channel on a second side ofthe plate; and first face piece to engage the first channel to define afirst fluid pathway; and a second face piece to engage the secondchannel to define a second fluid pathway, the first fluid pathway influid communication with the second fluid pathway and with the inletport and the outlet port.

In an example of the twenty-eighth aspect, the first and second facepiece comprise films.

In another example of the twenty-eighth aspect and the above examples,the film has a thickness of not greater than 1000 micrometers.

In an additional example of the twenty-eighth aspect and the aboveexamples, the film comprises a metal layer.

In another example of the twenty-eighth aspect and the above examples,the film is polymeric.

In a further example of the twenty-eighth aspect and the above examples,the device further includes a third channel on the first side of theplate and a fourth fluid channel on the second side of the plate, thethird and fourth channels cooperative with the first and second facepieces to define third and fourth fluid pathways in fluid communicationwith the first and second fluid pathways.

In a twenty-ninth aspect, a thermocycling system includes athermocycling device of the twenty-eighth aspect and associatedexamples; and a first heat plate in proximity to the first fluid pathwayand defining a first heating zone; and a second heat plate in proximityto the second fluid pathway and defining a second heating zone.

In a thirtieth aspect, a method of generating an amplified sampleincludes generating an emulsion from a carrier fluid and a sample fluidwith an emulsion generator. The emulsion generator includes a carrierfluid port to receive the carrier fluid, a sample fluid port to receivethe sample fluid, and an emulsion outlet port. The emulsion generatorincludes a membrane to generate an emulsion including the carrier fluidand the sample fluid. The emulsion exits the emulsion generator via theemulsion outlet port. The sample fluid includes conjugated beads. Themethod further includes heating the emulsion to an amplificationcondition with a heater of an amplification device. The amplificationdevice includes an inlet port in fluid communication with the emulsionoutlet port and to receive the emulsion. The amplification deviceincludes an effluent port. The amplified emulsion exits theamplification device via the effluent port. The amplified emulsionincludes amplified beads derived from the conjugated beads. The methodalso includes breaking the emulsion following heating with a centrifugedevice. The centrifuge device includes an inlet conduit in fluidcommunication with the effluent port and to receive the emulsion afterheating. The centrifuge device further includes a rotor to receive atube and a slinger to dispense the amplified emulsion received via theinlet conduit to the tube when the rotor is spinning The amplified beadsare dispensed to the tube.

In an example of the thirtieth aspect, the tube includes an extensionfluid passage extending from a mouth of the tube and radially outwardand the centrifuge further includes a peripheral gutter disposed aroundthe rotor and to receive fluid from the extension fluid passage of thetube. Breaking the emulsion includes dispensing the emulsion into theslinger when the rotor is spinning In another example of the thirtiethaspect and the above examples, the centrifuge further includes anadapter to receive the inlet conduit.

In a further example of the thirtieth aspect and the above examples, theadapter includes a casing including a central cavity and a carriagedisposed within the cavity. The carriage includes a bore to receive aneedle coupled to a terminal end of the inlet conduit. The adapterfurther includes a spring to motivate the carriage away from theslinger.

In an additional example of the thirtieth aspect and the above examples,the emulsion generator further includes a first channel gasket and asecond channel gasket disposed on opposite sides of the membrane.Generating the emulsion includes flowing the carrier fluid and thesample fluid through the membrane at least twice.

In another example of the thirtieth aspect and the above examples, theemulsion generator further includes a sample vial to couple to thesample fluid port and includes a displacement fluid inlet extending intoa sample vial coupled to the sample fluid port.

In a thirty-first aspect, an integrated apparatus includes an emulsiongenerator including a carrier fluid port to receive a carrier fluid, asample fluid port to receive a sample fluid immiscible with the carrierfluid, and an emulsion outlet port, the emulsion generator including amembrane to generate an emulsion including the carrier fluid and thesample fluid. The emulsion exits the emulsion generator via the emulsionoutlet port. The apparatus further includes an amplification deviceincluding an inlet port in fluid communication with the emulsion outletport and to receive the emulsion. The amplification device includes aheater to subject the emulsion to amplification conditions and includesan effluent port. The amplified emulsion exits the amplification devicevia the effluent port. The apparatus also includes a centrifuge deviceincluding an inlet conduit in fluid communication with the effluent portand to receive the amplified emulsion. The centrifuge device furtherincludes a rotor to receive a tube and including a slinger to dispensethe amplified emulsion received via the inlet conduit to the tube whenthe rotor is spinning

In an example of the thirty-first aspect, the tube includes an extensionfluid passage extending from a mouth of the tube and radially outward.In another example, the centrifuge further includes a peripheral gutterdisposed around the rotor and to receive fluid from the extension fluidpassage of the tube.

In a further example of the thirty-first aspect and the above examples,the centrifuge further includes an adapter to receive the inlet conduit.In an example, the adapter includes a casing including a central cavityand a carriage disposed within the cavity. The carriage includes a boreto receive a needle coupled to a terminal end of the inlet conduit. Inanother example, the adapter further includes a spring to motivate thecarriage away from the slinger. In an additional example, the casingfurther includes a wash fluid inlet.

In an additional example of the thirty-first aspect and the aboveexamples, the emulsion generator further includes a first channel gasketand a second channel gasket disposed on opposite sides of the membraneand defining a fluid path passing through the membrane at least twice.In an example, the fluid path is concurrent.

In another example of the thirty-first aspect and the above examples,the fluid path is countercurrent.

In a further example of the thirty-first aspect and the above examples,the emulsion generator further includes a sample vial to couple to thesample fluid port. In an example, the emulsion generator furtherincludes a displacement fluid inlet extending into a sample vial coupledto the sample fluid port.

In an additional example of the thirty-first aspect and the aboveexamples, the amplification device further includes an amplificationplate to receive the emulsion from the emulsion outlet port.

In another example of the thirty-first aspect and the above examples,the heater is to cycle the temperature of the amplification plate.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities, percentages orproportions, and other numerical values used in the specification andclaims, are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present invention. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a range of “less than 10” includes any and allsubranges between (and including) the minimum value of zero and themaximum value of 10, that is, any and all subranges having a minimumvalue of equal to or greater than zero and a maximum value of equal toor less than 10, as illustrated by the range of from 1 to 5.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed:
 1. A method of generating an amplified sample, themethod comprising: generating an emulsion from a carrier fluid and asample fluid with an emulsion generator, the emulsion generatorincluding a carrier fluid port to receive the carrier fluid, a samplefluid port to receive the sample fluid, and an emulsion outlet port, theemulsion generator including a membrane to generate an emulsionincluding the carrier fluid and the sample fluid, the emulsion exitingthe emulsion generator via the emulsion outlet port, the sample fluidincluding conjugated beads; heating the emulsion to an amplificationcondition with a heater of an amplification device, the amplificationdevice including an inlet port in fluid communication with the emulsionoutlet port and to receive the emulsion, the amplification deviceincluding an effluent port, the amplified emulsion exiting theamplification device via the effluent port, the amplified emulsionincluding amplified beads derived from the conjugated beads; andbreaking the emulsion following heating with a centrifuge device, thecentrifuge device including an inlet conduit in fluid communication withthe effluent port and to receive the emulsion after heating, thecentrifuge device further including a rotor to receive a tube and aslinger to dispense the amplified emulsion received via the inletconduit to the tube when the rotor is spinning, the amplified beadsbeing dispensed to the tube.
 2. The method of claim 1, wherein the tubeincludes an extension fluid passage extending from a mouth of the tubeand radially outward and wherein the centrifuge further includes aperipheral gutter disposed around the rotor and to receive fluid fromthe extension fluid passage of the tube, wherein breaking the emulsionincludes dispensing the emulsion into the slinger when the rotor isspinning
 3. The method of claim 1, wherein the centrifuge furtherincludes an adapter to receive the inlet conduit.
 4. The method of claim1, wherein the adapter includes a casing including a central cavity anda carriage disposed within the cavity, the carriage including a bore toreceive a needle coupled to a terminal end of the inlet conduit, theadapter further includes a spring to motivate the carriage away from theslinger.
 5. The method of claim 1, wherein the emulsion generatorfurther includes a first channel gasket and a second channel gasketdisposed on opposite sides of the membrane, wherein generating theemulsion includes flowing the carrier fluid and the sample fluid throughthe membrane at least twice.
 6. The method of claim 1, wherein theemulsion generator further includes a sample vial to couple to thesample fluid port and includes a displacement fluid inlet extending intoa sample vial coupled to the sample fluid port.
 7. An integratedapparatus comprising: an emulsion generator including a carrier fluidport to receive a carrier fluid, a sample fluid port to receive a samplefluid immiscible with the carrier fluid, and an emulsion outlet port,the emulsion generator including a membrane to generate an emulsionincluding the carrier fluid and the sample fluid, the emulsion exitingthe emulsion generator via the emulsion outlet port; an amplificationdevice including an inlet port in fluid communication with the emulsionoutlet port and to receive the emulsion, the amplification deviceincluding a heater to subject the emulsion to amplification conditionsand including an effluent port, the amplified emulsion exiting theamplification device via the effluent port; and a centrifuge deviceincluding an inlet conduit in fluid communication with the effluent portand to receive the amplified emulsion, the centrifuge device furtherincluding a rotor to receive a tube and including a slinger to dispensethe amplified emulsion received via the inlet conduit to the tube whenthe rotor is spinning
 8. The apparatus of claim 7, wherein the tubeincludes an extension fluid passage extending from a mouth of the tubeand radially outward.
 9. The apparatus of claim 8, wherein thecentrifuge further includes a peripheral gutter disposed around therotor and to receive fluid from the extension fluid passage of the tube.10. The apparatus of claim 7, wherein the centrifuge further includes anadapter to receive the inlet conduit.
 11. The apparatus of claim 10,wherein the adapter includes a casing including a central cavity and acarriage disposed within the cavity, the carriage including a bore toreceive a needle coupled to a terminal end of the inlet conduit.
 12. Theapparatus of claim 11, wherein the adapter further includes a spring tomotivate the carriage away from the slinger.
 13. The apparatus of claim11, wherein the casing further includes a wash fluid inlet.
 14. Theapparatus of claim 7, wherein the emulsion generator further includes afirst channel gasket and a second channel gasket disposed on oppositesides of the membrane and defining a fluid path passing through themembrane at least twice.
 15. The apparatus of claim 7, wherein the fluidpath is concurrent.
 16. The apparatus of claim 7, wherein the fluid pathis countercurrent.
 17. The apparatus of claim 7, wherein the emulsiongenerator further includes a sample vial to couple to the sample fluidport.
 18. The apparatus of claim 17, wherein the emulsion generatorfurther includes a displacement fluid inlet extending into a sample vialcoupled to the sample fluid port.
 19. The apparatus of claim 7, whereinthe amplification device further includes an amplification plate toreceive the emulsion from the emulsion outlet port.
 20. The apparatus ofclaim 7, wherein the heater is to cycle the temperature of theamplification plate.